Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab

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Aramini, Yves Aubin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4165568/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2024 Read the published version in Biomolecular NMR Assignments → Version 1 posted 7 You are reading this latest preprint version Abstract Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli . Monoclonal antibody trastuzumab NMR spectroscopy fragment antigen binding Escherichia coli Figures Figure 1 Figure 2 Figure 3 Figure 4 Biological context Human epidermal growth factor receptor type 2 (HER2) is a member of the superfamily of four receptor types (HER1, HER2, HER3, and HER-) involved in epithelial cell growth and differentiation. Ligand binding to the extracellular domain of the HER receptor induces the formation of homodimers or heterodimers, which in turn activates their intracellular tyrosine kinase domain. Then HER receptor will phosphorylate its dimer partner which will initiate a cascade of events leading to cell proliferation and differentiation(Patel et al., 2020a ). Amongst all receptors, HER2 is the only receptor type that has no known ligands, while it is the preferred dimerization partner with other HER types. Overexpression of HER2, is observed in 20–30% of early and advanced breast cancer mainly, but also in advanced stomach cancer and gastro-oesophageal junction cancer(Patel et al., 2020b ). In breast cancer cells, the high copy number of HER2 receptors can produce ligand-independent heterodimer formation with HER3 that activates this signaling pathway. HER2-positive cancers, especially breast cancer, have poor clinical prognosis. Trastuzumab (brand name Herceptin® and a number of biosimilars Herzuma®, Kanjinti®, Trazimera®, Ogivri®, Zercepac®, Trastucip®) is a therapeutic monoclonal antibody (mAb) that was developed to target HER2. It binds to the extracellular juxtamembrane domain of HER2 receptor to prevent the activation of its intracellular tyrosine kinase thereby inhibiting the proliferation and survival of HER2-dependent tumors(Hudis, 2007 ). Trastuzumab binding to HER2 inhibits the ligand-independent HER2-HER3 heterodimer formation and HER3 phosphorylation. This suppresses AKT (Protein kinase B) phosphorylation thereby deactivating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling(Yakes et al., 2002 ) pathway This pathway is highly activated in various types of cancer(Nicholson and Anderson, 2002 ; Carmona et al., 2016 ). Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of the anti-tumor function of trastuzumab which is mediated by effector immune cells such as natural killer cells (Kim et al., 2017 ; Tian et al., 2017 ). Binding of its fragment crystallizable gamma receptors to the antibody Fc fragment initiates the ADCC process leading to the destruction of tumor cells by the immune system. Trastuzumab is a humanized mAb of the immunoglobulin G1 (IgG1) class where the residues involved in antigen binding that form the complementary-determining region (CDR), are from mouse while all constant regions are from human IgG1. NMR methods at natural abundance have been proposed for the assessment of the higher order structure of mAbs and their Fab and Fc fragments(Brinson et al., 2019 ; Hodgson et al., 2019 ). However, the lack of resonance assignment limits the level of NMR characterization of these high molecular weight proteins (49–50 kDa for both Fab and Fc fragments). This has been impeded by the challenge of producing isotopically labelled, in particular highly deuterated, mAb fragments. Recently, that limit has been crossed. Solomon and coworkers reported the backbone resonance assignment of yeast produced NIST-mAb Fab(Chao et al., 2023 ; Solomon et al., 2023 ). However, more complex labelling schemes with yeast, such as methyl labelling, are not straightforward and thus require further developments. In parallel, we developed a method using Escherichia coli to produce isotopically labelled mAb Fab fragments for NMR resonance assignment (Gagné et al., 2023 ). The method, is based on the production of a single polypeptide chain in inclusion bodies constructed by the fusion of the heavy and the light chains with a removable linker to facilitate protein refolding. Production of labelled Fab fragments using this method afforded many advantages. All amide deuterons are readily exchanged with protons during the refolding procedure. E. coli allows easy isotope incorporation and various labelling schemes such as methyl labelling and surprisingly higher protein yields of 99% deuterated samples. Here we present the backbone and side chain methyl group of isoleucine, leucine and valine residues chemical shifts, including the stereo assignments of leucine and valine methyl groups, of the single chain Fab fragment of trastuzumab. Methods and experiments Expression and purification of trastuzumab-scFab The amino acid sequence of used for the production of samples of labelled trastuzumab-scFab has been described previously (Gagné et al., 2023 ). The heavy chain of the Fab domain, residues Glu1 to Pro230 (underlined) where Cys229 has been mutated to Ala229, was linked to residues Asp1 to Cys214 of the light chain via a linker made of five (GGGGS) elements plus SSGLVPRGS. The last residues of the linker contain a thrombin recognition site (LVPRGS). A poly-histidine tag (MGSSHHHHHH HHHHSSGHMLVPR) is fused to the amino terminal of this polypeptide. Thrombin cleavage only removed the fusion tag leaving the linker intact. No attempts were made to cleave the linker with papain post-thrombin cleavage of the tag. We therefore elected to carry out the assignment of trastuzumab-scFab fragment with the following sequence: 1-GS EVQLVESG GGLVQPGGSL RLSCAASGFN IKDTYIHWVR QAPGKGLEWV ARIYPTNGYT 61- RYADSVKGRF TISADTSKNT AYLQMNSLRA EDTAVYYCSR WGGDGFYAMD YWGQGTLVTV 121- SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 180- SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT AP GGGGSGGG 241- GSGGGGSGGG GSGGGGSGGG GSSSGLVPRG S DIQMTQSPS SLSASVGDRV TITCRASQDV 301- NTAVAWYQQK PGKAPKLLIY SASFLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ 361- QHYTTPPTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK 421- VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF 481- NRGEC Expression of labeled 2 H- 13 C- 15 N-trastuzumab-scFab was carried out by incubating Escherichia coli BL21(DE3) harboring the Histag-trastuzumab-scFab construct (Gagné et al., 2023 ) at 37℃ (225 rpm) in M9/D 2 O minimal media supplemented with 2 g/L 2 H- 13 C -glucose and 3 g/L 15 N-ammonium chloride as sole source of carbon and nitrogen. Briefly, one colony was transferred to 4 mL of Luria Broth Miller (LB) and incubated for 2h at 37℃ (225 rpm). A 500 µL aliquot of the LB pre-culture was transferred to 50 mL of M9/H 2 O for another 6h of incubation after which, 2 mL was transferred into 200 mL of M9/D 2 O for an overnight pre-culture. The following day, the content was transferred to 2 liters of M9/D 2 O and returned to the incubator. Induction with 1 mM thio-D-galactopyranoside (IPTG) was conducted when the OD 600 was 0.67. After 24h of expression, cells were recovered by centrifugation at 3,011 x g and stored at -80℃ until used. Expression of labeled 2 H- 13 C- 15 N- 1 H-methyl-(Ile, Leu, Val)-trastuzumab-scFab was conducted as described above, with the exception that 25 mg of α-ketobutyric acid- 13 C-3,3-d 2 and 50 mg of α-ketoisovaleric-U- 13 C 5 acid-3-d 1 (dry powder) were added directly into the culture at an OD 600 of 0.38, while the induction was conducted with 1 mM IPTG at an OD 600 of 0.77. Fractionally labeled 13 C(10%)- 15 N-trastuzumab-scFab was obtained by expressing the protein in a mixture of 10% 13 C6 glucose and 90% un-labeled glucose as the sole carbon source. Protein purifications were conducted using a fast dilution approach at pH 9.0 and with 2 M L-arginine, as described in previously (Gagné et al., 2023 ). His tag was removed by incubating the protein with 100 units/mg of thrombin in phosphate buffer at pH 7.0 for 5h at 37℃ under light agitation. Thrombin and Histag were removed with a Hitrap SP (5 mL), followed by size exclusion chromatography using HiLoad 26/60 Superdex 75 pg. Protein yield (before cleavage) was 37, 44, and 36 mg/L of culture were obtained for 2 H- 13 C- 15 N-trastuzumab-scFab, 1 H-I(δ1)LVmethyl- 2 H- 13 C- 15 N-trastuzumab-scFab, and 1 H- 13 C(10%)- 15 N-trastuzumab-scFab, respectively. Sample for resonance assignment contained 395 µM 2 H- 13 C- 15 N-trastuzumab-scFab (21 mg/mL) in 20 mM sodium acetate-d 3 at pH 5.0 with 5% v/v deuterium oxide for lock frequency purposes in 50 µL and transferred in a 1.7 mm tube. Sample for side-chain methyl groups, prepared as described above with the addition of labelled intermediates as described by Goto and coworkers (Goto et al., 1999 ), contained 400 µM 1 H-I(δ1)LVmethyl- 2 H- 13 C- 15 N-trastuzumab-scFab in 300 µL (5 mm Shigemi tube) in the same buffer. Data were collected at 40℃ (313 K) on Bruker AVANCE NEO 600 MHz (side-chain assignment), AVANCE III-HD 700 MHz (backbone assignment) and AVANCE NEO 1GHz NMR spectrometers equipped with 5mm, 1.7mm, and 5mm, respectively, TCI cryogenically cooled triple resonance inverse probeheads fitted with z-axis gradients. Chemical shift resonances were referenced with sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). NMR experiments Data collection for the assignment of the backbone resonances used the TROSY-based version(Eletsky et al., 2001 ) of the standard pulse sequences with deuterium decoupling during carbon evolution from the Bruker library: 2D- 15 NHSQC (trosyetf3gpsi), 3D-HNCO (trhncogp2h3d) (Salzmann et al., 1998 ), 3D-HN(CA)CO(Clubb and Wagner, 1992 ), 3D-HNCA (trhncagp2h3d2), 3D-HN(CO)CA (trhncocagp2h3d) (Eletsky et al., 2001 ), 3D-HNCACB (trhncacbgp2h3d), 3D-HN(CO)CACB (trhncocacbgp2h3d) (Grzesiek and Bax, 1993 ; Eletsky et al., 2001 ) on the 700 MHz NMR spectrometer fitted with the 1.7 mm NMR probehead. All proton-nitrogen planes were collected using a spectral width (SW) of 18 ppm with 2048 real points ( 1 H) and 40 ppm with 64 real points ( 15 N). The 13 C indirect dimensions were collected with a SW of 14 ppm, and 128 real points for HNCO/HNCACO, a SW of 30 ppm with 128 real points for HNCA/NHCOCA, and a SW of 80 ppm with 128 real points for HNCACB/HNCOCACB. The acquisition time of 13 h (46 h) for the HNCO (TROSY) and 52 h for all other 3D experiments was used. Side-chain methyl resonance of isoleucines (delta), leucines and valines were assigned with pulse sequences based on a carbon TOCSY element to transfer the methyl carbon magnetization down to either the alpha carbon or carbonyl prior to transfer to the bonded nitrogen and then proton for detection. These experiments are most efficient at fields of 600 MHz or less. Data were collected on a sample of 1 H-I(δ1)LVmethyl- 2 H- 13 C- 15 N-trastuzumab-scFab at 465 µM (25 mg/mL) in 20 mM sodium acetate-d3 at pH 5.0 with 5% v/v deuterium oxide, in a 5 mm Shigemi tube. NMR pulse sequence codes were graciously provided by Prof. Lewis Kay (University of Toronto). In total a series of four 3D experiments 3D-CCC(CO)NH, 3D-HCC(CO)NH, 3D-CCC(CA)NH, and 3D-HCC(CA)NH were acquired (Tugarinov and Kay, 2003 ). Data were collected with a SW of 16 ppm with 2048 real points in the proton direct dimension and a SW of 40 ppm with 64 real points in the nitrogen dimension centered at 120 ppm. The indirect proton dimensions (HCC-) were collected with a SW of 3 ppm with 64 real points and the indirect carbon dimensions (CCC-) with a SW of 22 ppm with 54 real points for a total acquisition time of 54 h and 64 h, respectively. Stereoassignment of the Pro-R and Pro-S of methyl groups of valine and leucine side chains was carried out following the method of Neri et al (Neri et al., 1989 ). Briefly, a constant time 2D- 1 H- 13 C CT-HSQC was recorded on a 1 H- 13 C(10%)- 15 N-trastuzumab-scFab sample using a constant time of 28 ms during carbon evolution. The resulting spectrum provided resonances of opposite phases for the Pro-R and Pro-S methyl groups (Tugarinov and Kay, 2004 ). Data analysis and resonance assignment and validation NMR data were processed using nmrPipe (Delaglio et al., 1995 ) that was run via the NMRBox web facility (Maciejewski et al., 2017 ) and visualized with NMRViewJ (Johnson and Blevins, 1994 ). Semi-automated sequential assignment was carried out with the Runabout tool of NMRViewJ software. Validation of NMR assignment The web server of I-PINE ( http://i-pine.nmrfam.wisc.edu/index.html ) (Lee et al., 2019 ) was used to verify and validate the trastuzumab-scFab assignments. All peak lists from TROSY-based NMR experiments, namely 2D- 15 N-HSQC, 3D-HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB, were used as input files supplemented with the Runabout-manual assignment list and the three-dimensional X-ray structure (PDB ID 5xhg). The output of the server allowed the identification and correction of a few assignments, the identification of new assignments. In addition, we tested the new assignment protocol BARASA (Bishop et al., 2023 ) running under NMRBox using the same peak list and assignments as input data used for I-PINE. A total of 20 runs were conducted, using 80 concurrent threads with 0.99 convergence p-value, and a stepwise energy drop of -100. Ca, Cb, and CO zero points were all set to 0.20, with a chemical shift energy range of -50 to 100. Secondary structure predictions based on using 13 Ca, 13 Cb, 13 CO, and 15 N chemical shift resonances of 2 H- 13 C- 15 N-trastuzumab-scFab were performed using the web server of CIS 3.0 (Hafsa et al., 2015 ) ( http://csi3.wishartlab.com/cgi-bin/index.php ) and TALOS-N (Bartels et al., 1996 ) inside NMRBox (Maciejewski et al., 2017 ). Secondary structure elements predicted from chemical shifts were compared to the X-ray structure (PDB ID 5xhg). The validated assignments were deposited in the BioMagResBank under accession number 52228. Results Resonance assignment of backbone atoms Attempts to remove the linker after thrombin cleavage of the fused poly-histidine tag yielded significant loss of sample. Therefore, we compared the 2D- 1 H- 15 N HSQC of trastuzumab-scFab with the fully cleaved trastuzumab-Fab. The extra resonances belonging to the linker (scFab) were well resolved from any backbone resonances of the Fab and all backbone resonances of both samples were overlapped, indicating that the assignment of the scFab can be directly used for the Fab ( Fig 1 ). The fragment contained a total of 485 amino acids (50.8 kDa) with the heavy having 230 residues (13 prolines) and the light having 214 residues (12 prolines). The 2 H- 13 C- 15 N two-dimensional TROSY HSQC spectrum of trastuzumab-scFab shows well-dispersed resonances, typical of well-folded Fab ( Fig 2 ). A total of 405 (96.7%) 1 H- 15 N backbone peaks were assigned, with 204 (94.0%) and 201 (99.5%) in the heavy and light chains, respectively. Assigned carbons include 416 (93.7%) 13 CO, 422 (95.0%) 13 Ca, and 373 (91.0%) non-glycine 13 Cb. From the 41 residues containing linker, the first two glycines were assigned and the last seven residues including the thrombin site. The Fab fragment is composed of four immunoglobulin domains that are each stabilized by one disulfide bond: Cys24-Cys98 (heavy chain, VH), Cys149-Cys205 (heavy chain, CH1), Cys294-Cys359 (light chain, VL), Cys405-Cys465 (light chain, CL), and one bond that links the heavy to the light chain Cys225-Cys485. All cysteine Cb chemical shifts are higher than 35 ppm, which is indicative of properly formed disulfide bonds, while reduced cysteine would have chemical shifts less than 35 ppm (Schulte et al. , 2020). Resonance assignment of isoleucine delta-1, leucine and valine methyl groups Using the carbon TOCSY versions of experiments, a total of 11 isoleucines (100%), 28 leucines (93%), and 35 valines (92%) were assigned ( Fig 3 ). Only four residues have not been assigned, namely Leu20, Leu198, Val93, Val193. Analysis of the 2D 1 H- 13 C constant time HMQC experiment on a 10% 13 C-labeled (90% natural abundance) sample, provided complete stereoassignment for all 28 leucine and 35 valine methyl groups. We used the 1983 IUPAC-IUC recommendation for the identification of the stereospecificity of methyl groups, with Pro-R and Pro-S being identified as g1 or d1, and g2 or d2 for valine and leucine, respectively (Markley et al. , 1998). Comparison with NISTmAb assignment Trastuzumab and the NIST-mAb are two monoclonal antibodies of the IgG1 class with light chain kappa. They both share identical primary sequences in their constant heavy 1 (C H 1) and constant light domain (C L ). In order to further validate our assignment, we compared it to the resonance assignment of backbone atoms of the NISTmAb-Fab(Solomon et al. , 2023). It is expected that resonances arising from amides with the same local magnetic environment will have the same chemical shifts while others that have similar or slightly different environments will produce slightly or significantly different chemical shifts. Indeed, comparison of assigned amide groups with same or very similar chemical shifts from both mAbs yielded the same assignment. Validation of the backbone assignment I-PINE was used to validate the current assignment and to help in identifying non-assigned residues. From a total of 419 non-proline residues, I-PINE assigned 403 (96.21%) residues thus providing 15 new assignments. From this total, 370 (95.4%) matched our assignment. The Linear Analysis of Chemical Shifts (LACS) identified 2 outliers: Ser19 in 13 C-O, and Val95 in 13 Ca and 13 Cb. All cysteines are fully oxidized, with the exception of Cys98, with 74.9% oxidation. Prolines' isomerization state is mostly trans, with the exception of Pro158, Pro279, and Pro412. Predominantly trans prolines are associated with folded proteins (Alderson et al. , 2018). Initial attempts using BARASA using default parameters to validate the assignment led to poor results: only 195 (46.5%) residues out of the 419 were identified (Table 1). However, optimization of the parameters increased the number of assigned residues to 361 (86.2%), corresponding to 77.4% and 95.5% of the residues of the heavy and light chains, respectively. While BARASA did not provide a higher number of assigned residues, the approach did allow the identification of errors or glitches in the semi-automatic assignment performed with RunAbout such as misinterpretation of which resonance belonged to the Cb(i) vs Cb( i-1) etc. Table 1: Parameters optimization in BARASA. Stepwise energy drop -100 -500 -1000 -2000 Convergence p-value: 0.99; Min. chemical shift energy: -50 No. of assignments 1 361 351 333 328 Matching assignments 2 335 (92.8%) 326 (92.9%) 312 (93.7%) 309 (94.2%) Convergence p-value 0.50 0.99 Stepwise energy drop: -2000; Min. chemical shift energy: -50 No. of assignments 1 195 328 Matching assignments 2 187 (95.9%) 309 (94.2%) Min chem. shift energy -50 -100 Stepwise energy drop: -2000; Convergence p-value: 0.99 No. of assignments 1 328 344 Matching assignments 2 309 (94.2%) 318 (92.4%) Note: Default parameters: stepwise energy drop: -2000; convergence p-value: 0.99; Min. chemical shift energy: -50 1. The number of assignments corresponds to the results returned from the calculations 2. Matching assignment between BMRB 52228 and the results returned from BARASA In order to test the ability of BARASA to perform de novo resonance assignment of trastuzumab-Fab, we used a SHIFTX2 predicted list of backbone chemical shifts and a mix of ‘known’ and predicted chemical shifts. The list of known chemical shifts were built from residues of the constant domains C H 1 and C L of trastuzumab-Fab and NIST-Fab that shares the same chemical shifts. All remaining (unknown) residues were predicted with SHIFTX2. Finally, a test with SHIFTX2 predicted chemical shifts was carried out. The results (Table 2) showed that both approaches produced very good assignments (over 88%) with a very good assignment of the light chain (>90%). Both procedures obtained lower assignments on the heavy chain, similar to the manual-semi-automatic assignment. This region of the Fab domain showed lower spectral resolution and was more challenging. Table 2: Test of de novo assignments using BARASA using the above the following parameters: convergence p-value: 0.99; Stepwise energy drop: -100; Min. chemical shift energy: -50 Pre-assign List RunAbout MIX SHIFTX2 No. of assignments 1 361 370 335 Matching assignments 2 335/361 (92.8%) 328/370 (88.6%) 304/335 (90.7%) Matching HC-Variable (117) 3 73/81 (90.1%) 68/93 (73.1%) 67/83 (80.7%) Matching HC-Constant (100) 3 80/87 (92.0%) 78/87 (89.7%) 66/69 (95.7%) Matching LC-Variable (102) 3 90/94 (95.7%) 88/92 (95.7%) 86/94 (91.5%) Matching LC-Constant (101) 3 92/99 (92.9%) 94/98 (95.9%) 85/89 (95.5%) Notes: total number of non-proline residues (excluding the linker) =420 1. The number of assignments corresponds to the results returned from the calculations 2. Matching assignment between BMRB 52228 and the results returned from BARASA 3. Matching assignment between BMRB 52228 and corresponding domains: V H , C H 1, V L , and C L Prediction of secondary structure elements using CIS3.0 and TALOS-N allowed further validation of the assignments. Predicted values of backbone torsion angles, visualized as secondary structure elements, are consistent with the X-ray structure (PDB ID 5xhg) ( Fig 4 ). Abbreviations mAb: monoclonal antibody. scFab: single-chain fragment antigen-binding. Fab: fragment antigen-binding VH: heavy chain variable domain. VL: light chain variable domain. Declarations Ethics approval and consent to participate Not applicable Consent for publication The authors declare that they have no conflict of interest. Availability of data and material Chemical shifts and Bruker raw data ser files were deposited in the BMRB data bank with entry number 52228. Competing interests The authors declare that they have no conflict of interest. Funding Not applicable Authors' contributions Conceptualization: Yves Aubin; Investigation: Donald Gagné, James Aramini; Formal Analysis: Donald Gagné, James Aramini, Visualization: Donald Gagné, Yves Aubin; Writing-original draft: Donald Gagné, Yves Aubin; Writing-review and editing: Donald Gagné, Yves Aubin; Supervision: Yves Aubin. Acknowledgements We thank Drs. Michael Johnston and Huixin Lu for critical reading of the manuscript. References ALDERSON, T. R., LEE, J. H., CHARLIER, C., YING, J. & BAX, A. 2018. Propensity for cis-Proline Formation in Unfolded Proteins. Chembiochem, 19 , 37-42. DOI: 10.1002/cbic.201700548. BARTELS, C., BILLETER, M., GUNTERT, P. & WUTHRICH, K. 1996. Automated sequence-specific NMR assignment of homologous proteins using the program GARANT. J Biomol NMR, 7 , 207-13. DOI: 10.1007/BF00202037. BISHOP, A. C., TORRES-MONTALVO, G., KOTARU, S., MIMUN, K. & WAND, A. J. 2023. 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NMRbox: A Resource for Biomolecular NMR Computation. Biophys J, 112 , 1529-1534. DOI: 10.1016/j.bpj.2017.03.011. MARKLEY, J. L., BAX, A., ARATA, Y., HILBERS, C. W., KAPTEIN, R., SYKES, B. D., WRIGHT, P. E. & WUTHRICH, K. 1998. Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. Eur J Biochem, 256 , 1-15. DOI: 10.1046/j.1432-1327.1998.2560001.x. NERI, D., SZYPERSKI, T., OTTING, G., SENN, H. & WUTHRICH, K. 1989. Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry, 28 , 7510-6. DOI: 10.1021/bi00445a003. NICHOLSON, K. M. & ANDERSON, N. G. 2002. The protein kinase B/Akt signalling pathway in human malignancy. Cellular Signalling, 14 , 381-395. DOI: https://doi.org/10.1016/S0898-6568(01)00271-6. PATEL, A., UNNI, N. & PENG, Y. 2020a. The Changing Paradigm for the Treatment of HER2-Positive Breast Cancer. Cancers [Online], 12. PATEL, A., UNNI, N. & PENG, Y. 2020b. The Changing Paradigm for the Treatment of HER2-Positive Breast Cancer. . Cancers(Basel), 12 , 2081-2097. DOI: 10.3390/cancers12082081. SALZMANN, M., PERVUSHIN, K., WIDER, G., SENN, H. & WUTHRICH, K. 1998. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci U S A, 95 , 13585-90. DOI: 10.1073/pnas.95.23.13585. SCHULTE, L., MAO, J., REITZ, J., SREERAMULU, S., KUDLINZKI, D., HODIRNAU, V. V., MEIER-CREDO, J., SAXENA, K., BUHR, F., LANGER, J. D., et al. 2020. Cysteine oxidation and disulfide formation in the ribosomal exit tunnel. Nat Commun, 11 , 5569. DOI: 10.1038/s41467-020-19372-x. SOLOMON, T. L., CHAO, K., GINGRAS, G., AUBIN, Y., O’DELL, W. B., MARINO, J. P. & BRINSON, R. G. 2023. Backbone NMR assignment of the yeast expressed Fab fragment of the NISTmAb reference antibody. Biomolecular NMR Assignments . DOI: 10.1007/s12104-023-10123-9. TIAN, X., WEI, F., WANG, L., YU, W., ZHANG, N., ZHANG, X., HAN, Y., YU, J. & REN, X. 2017. Herceptin Enhances the Antitumor Effect of Natural Killer Cells on Breast Cancer Cells Expressing Human Epidermal Growth Factor Receptor-2. Front Immunol, 8 , 1426. DOI: 10.3389/fimmu.2017.01426. TUGARINOV, V. & KAY, L. E. 2003. Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc, 125 , 13868-78. DOI: 10.1021/ja030345s. TUGARINOV, V. & KAY, L. E. 2004. Stereospecific NMR assignments of prochiral methyls, rotameric states and dynamics of valine residues in malate synthase G. J Am Chem Soc, 126 , 9827-36. DOI: 10.1021/ja048738u. YAKES, F. M., CHINRATANALAB, W., RITTER, C. A., KING, W., SEELIG, S. & ARTEAGA, C. L. 2002. Herceptin-induced Inhibition of Phosphatidylinositol-3 Kinase and Akt Is Required for Antibody-mediated Effects on p27, Cyclin D1, and Antitumor Action1. Cancer Research, 62 , 4132-4141. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 May, 2024 Read the published version in Biomolecular NMR Assignments → Version 1 posted Editorial decision: Revision requested 19 Apr, 2024 Reviews received at journal 19 Apr, 2024 Reviewers agreed at journal 01 Apr, 2024 Reviewers invited by journal 01 Apr, 2024 Submission checks completed at journal 26 Mar, 2024 Editor assigned by journal 26 Mar, 2024 First submitted to journal 25 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4165568","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284016887,"identity":"7080f94e-2064-41d2-8667-fb2f2c073cd9","order_by":0,"name":"Donald Gagné","email":"","orcid":"","institution":"Health Canada","correspondingAuthor":false,"prefix":"","firstName":"Donald","middleName":"","lastName":"Gagné","suffix":""},{"id":284016888,"identity":"e7f077a8-0731-4874-aced-f7cc34f015ba","order_by":1,"name":"James M. Aramini","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"M.","lastName":"Aramini","suffix":""},{"id":284016889,"identity":"72e3e9b2-713a-4172-933f-a145b528502f","order_by":2,"name":"Yves Aubin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3RsWvCQBTH8V84eJN465MI6Z8Qlyz+MxEhXayLq4jTTVfn7v0fdI0c6BJx7RgpZA4UQqZQI4UOJUfdCr3veNwH7t0DXK4/XEwQh7sJJXcSoBf97mr4NjUllmbe93WV11gFkK856qWNJAnjYBY0PO1GGma05iL0tGWuMMuu7yEzUfy09b0mjcEZBMhGzh8lmpbMCh9YfZHGQo4a7KkboSsRMaSG8FQ3GRxVxJPN44I4iQbtLIop3D9vuknfiPeyrMbz4GVacPtjUppLXlfd5CHFbSnfEcdIuwEQrH8cSStwuVyuf9gnHIxLW/7wSSAAAAAASUVORK5CYII=","orcid":"","institution":"Health Canada","correspondingAuthor":true,"prefix":"","firstName":"Yves","middleName":"","lastName":"Aubin","suffix":""}],"badges":[],"createdAt":"2024-03-25 20:46:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4165568/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4165568/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12104-024-10177-3","type":"published","date":"2024-05-08T03:58:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53723930,"identity":"c6ed07be-637d-4dd1-be4f-1a0d0e39e009","added_by":"auto","created_at":"2024-03-29 11:20:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":690007,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of trastuzumab-scFab (red, thrombin cleaved) and trastuzumab-Fab (blue, papain cleaved).\u0026nbsp; The 2D-\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN-SOFAST-HMQC spectrum overlay recorded at 50ºC indicates that all backbone resonances of the scFab (linker present).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4165568/v1/11079fd71124479e38b4aa5a.png"},{"id":53723932,"identity":"cc91270b-ec20-4458-99db-f153fe197d33","added_by":"auto","created_at":"2024-03-29 11:20:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1106355,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN-TROSY-HSQC spectrum of \u003csup\u003e1\u003c/sup\u003eH-I(δ1)LVmethyl-\u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab acquired at 1 GHz at 40℃.\u0026nbsp; The backbone amide chemical shift assignment of heavy and light chains is shown in blue and orange, respectively.\u0026nbsp; The low field region of the spectrum is shown in the bottom-right box.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4165568/v1/831e86620bb878f43e006675.png"},{"id":53723931,"identity":"e2b51aef-226d-40c4-aafe-ec7667bb4346","added_by":"auto","created_at":"2024-03-29 11:20:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":409827,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC CT-HSQC spectrum of the methyl spectral region of trastuzumab-scFab.\u0026nbsp; The spectrum was acquired using a \u003csup\u003e1\u003c/sup\u003eH-I(d)LVmethyl-\u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab sample, with acquisition at 40℃ and 1 GHz, with 28 ms constant-time delay.\u0026nbsp; The methyl chemical shift assignment of isoleucine-d1, leucine, and valine of heavy and light chains is shown in blue and orange, respectively.\u0026nbsp; Stereospecific NMR assignment of the valines and leucines was conducted with 10% \u003csup\u003e13\u003c/sup\u003eC and 90% unlabeled carbon as the sole carbon source.\u0026nbsp; Pro-R (g1 of valine and d1 of leucine) and Pro-S (g2 of valine and d2 of leucine) were determined using 14 and 21 ms of constant-time delays\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4165568/v1/e4131cca0f9903014fbcc86b.png"},{"id":53723934,"identity":"75a546fd-454b-4f31-8cc1-9101e45513a7","added_by":"auto","created_at":"2024-03-29 11:20:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1291304,"visible":true,"origin":"","legend":"\u003cp\u003ePredictions of secondary structure elements of trastuzumab-scFab from backbone chemical shift using web platforms CIS 3.0 and TALOS-N compared with the X-ray structure. Helices are labeled H (red), beta-strands are labeled B (cyan) and coils are labeled C (grey).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4165568/v1/40ea345d8650546fbed5d3f6.png"},{"id":56140493,"identity":"eadbfb51-862e-4eb9-a4ed-276218e63d07","added_by":"auto","created_at":"2024-05-09 04:28:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2070761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4165568/v1/031fb6d4-f578-442a-a096-ff7845d87287.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab","fulltext":[{"header":"Biological context","content":"\u003cp\u003eHuman epidermal growth factor receptor type 2 (HER2) is a member of the superfamily of four receptor types (HER1, HER2, HER3, and HER-) involved in epithelial cell growth and differentiation. Ligand binding to the extracellular domain of the HER receptor induces the formation of homodimers or heterodimers, which in turn activates their intracellular tyrosine kinase domain. Then HER receptor will phosphorylate its dimer partner which will initiate a cascade of events leading to cell proliferation and differentiation(Patel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Amongst all receptors, HER2 is the only receptor type that has no known ligands, while it is the preferred dimerization partner with other HER types. Overexpression of HER2, is observed in 20\u0026ndash;30% of\u003c/p\u003e \u003cp\u003eearly and advanced breast cancer mainly, but also in advanced stomach cancer and gastro-oesophageal junction cancer(Patel et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). In breast cancer cells, the high copy number of HER2 receptors can produce ligand-independent heterodimer formation with HER3 that activates this signaling pathway. HER2-positive cancers, especially breast cancer, have poor clinical prognosis. Trastuzumab (brand name Herceptin\u0026reg; and a number of biosimilars Herzuma\u0026reg;, Kanjinti\u0026reg;, Trazimera\u0026reg;, Ogivri\u0026reg;, Zercepac\u0026reg;, Trastucip\u0026reg;) is a therapeutic monoclonal antibody (mAb) that was developed to target HER2. It binds to the extracellular juxtamembrane domain of HER2 receptor to prevent the activation of its intracellular tyrosine kinase thereby inhibiting the proliferation and survival of HER2-dependent tumors(Hudis, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Trastuzumab binding to HER2 inhibits the ligand-independent HER2-HER3 heterodimer formation and HER3 phosphorylation. This suppresses AKT (Protein kinase B) phosphorylation thereby deactivating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling(Yakes et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) pathway This pathway is highly activated in various types of cancer(Nicholson and Anderson, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Carmona et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of the anti-tumor function of trastuzumab which is mediated by effector immune cells such as natural killer cells (Kim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Binding of its fragment crystallizable gamma receptors to the antibody Fc fragment initiates the ADCC process leading to the destruction of tumor cells by the immune system. Trastuzumab is a humanized mAb of the immunoglobulin G1 (IgG1) class where the residues involved in antigen binding that form the complementary-determining region (CDR), are from mouse while all constant regions are from human IgG1.\u003c/p\u003e \u003cp\u003eNMR methods at natural abundance have been proposed for the assessment of the higher order structure of mAbs and their Fab and Fc fragments(Brinson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hodgson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the lack of resonance assignment limits the level of NMR characterization of these high molecular weight proteins (49\u0026ndash;50 kDa for both Fab and Fc fragments). This has been impeded by the challenge of producing isotopically labelled, in particular highly deuterated, mAb fragments. Recently, that limit has been crossed. Solomon and coworkers reported the backbone resonance assignment of yeast produced NIST-mAb Fab(Chao et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Solomon et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, more complex labelling schemes with yeast, such as methyl labelling, are not straightforward and thus require further developments. In parallel, we developed a method using \u003cem\u003eEscherichia coli\u003c/em\u003e to produce isotopically labelled mAb Fab fragments for NMR resonance assignment (Gagn\u0026eacute; et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The method, is based on the production of a single polypeptide chain in inclusion bodies constructed by the fusion of the heavy and the light chains with a removable linker to facilitate protein refolding. Production of labelled Fab fragments using this method afforded many advantages. All amide deuterons are readily exchanged with protons during the refolding procedure. \u003cem\u003eE. coli\u003c/em\u003e allows easy isotope incorporation and various labelling schemes such as methyl labelling and surprisingly higher protein yields of 99% deuterated samples. Here we present the backbone and side chain methyl group of isoleucine, leucine and valine residues chemical shifts, including the stereo assignments of leucine and valine methyl groups, of the single chain Fab fragment of trastuzumab.\u003c/p\u003e"},{"header":"Methods and experiments","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExpression and purification of trastuzumab-scFab\u003c/h2\u003e \u003cp\u003eThe amino acid sequence of used for the production of samples of labelled trastuzumab-scFab has been described previously (Gagn\u0026eacute; et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The heavy chain of the Fab domain, residues Glu1 to Pro230 (underlined) where Cys229 has been mutated to Ala229, was linked to residues Asp1 to Cys214 of the light chain via a linker made of five (GGGGS) elements plus SSGLVPRGS. The last residues of the linker contain a thrombin recognition site (LVPRGS). A poly-histidine tag (MGSSHHHHHH HHHHSSGHMLVPR) is fused to the amino terminal of this polypeptide. Thrombin cleavage only removed the fusion tag leaving the linker intact. No attempts were made to cleave the linker with papain post-thrombin cleavage of the tag. We therefore elected to carry out the assignment of trastuzumab-scFab fragment with the following sequence:\u003c/p\u003e \u003cp\u003e1-GS\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eEVQLVESG GGLVQPGGSL RLSCAASGFN IKDTYIHWVR QAPGKGLEWV ARIYPTNGYT\u003c/span\u003e\u003c/p\u003e \u003cp\u003e61-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eRYADSVKGRF TISADTSKNT AYLQMNSLRA EDTAVYYCSR WGGDGFYAMD YWGQGTLVTV\u003c/span\u003e\u003c/p\u003e \u003cp\u003e121-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ\u003c/span\u003e\u003c/p\u003e \u003cp\u003e180-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT AP\u003c/span\u003e\u003cb\u003eGGGGSGGG\u003c/b\u003e\u003c/p\u003e \u003cp\u003e241-\u003cb\u003eGSGGGGSGGG GSGGGGSGGG GSSSGLVPRG S\u003c/b\u003e\u003cem\u003eDIQMTQSPS SLSASVGDRV TITCRASQDV\u003c/em\u003e\u003c/p\u003e \u003cp\u003e301-\u003cem\u003eNTAVAWYQQK PGKAPKLLIY SASFLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ\u003c/em\u003e\u003c/p\u003e \u003cp\u003e361-\u003cem\u003eQHYTTPPTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK\u003c/em\u003e\u003c/p\u003e \u003cp\u003e421-\u003cem\u003eVDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF\u003c/em\u003e\u003c/p\u003e \u003cp\u003e481-\u003cem\u003eNRGEC\u003c/em\u003e\u003c/p\u003e \u003cp\u003eExpression of labeled \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab was carried out by incubating \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3) harboring the Histag-trastuzumab-scFab construct (Gagn\u0026eacute; et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) at 37℃ (225 rpm) in M9/D\u003csub\u003e2\u003c/sub\u003eO minimal media supplemented with 2 g/L \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC -glucose and 3 g/L \u003csup\u003e15\u003c/sup\u003eN-ammonium chloride as sole source of carbon and nitrogen. Briefly, one colony was transferred to 4 mL of Luria Broth Miller (LB) and incubated for 2h at 37℃ (225 rpm). A 500 \u0026micro;L aliquot of the LB pre-culture was transferred to 50 mL of M9/H\u003csub\u003e2\u003c/sub\u003eO for another 6h of incubation after which, 2 mL was transferred into 200 mL of M9/D\u003csub\u003e2\u003c/sub\u003eO for an overnight pre-culture. The following day, the content was transferred to 2 liters of M9/D\u003csub\u003e2\u003c/sub\u003eO and returned to the incubator. Induction with 1 mM thio-D-galactopyranoside (IPTG) was conducted when the OD\u003csub\u003e600\u003c/sub\u003e was 0.67. After 24h of expression, cells were recovered by centrifugation at 3,011 x g and stored at -80℃ until used.\u003c/p\u003e \u003cp\u003eExpression of labeled \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e1\u003c/sup\u003eH-methyl-(Ile, Leu, Val)-trastuzumab-scFab was conducted as described above, with the exception that 25 mg of α-ketobutyric acid-\u003csup\u003e13\u003c/sup\u003eC-3,3-d\u003csub\u003e2\u003c/sub\u003e and 50 mg of α-ketoisovaleric-U-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e5\u003c/sub\u003e acid-3-d\u003csub\u003e1\u003c/sub\u003e (dry powder) were added directly into the culture at an OD\u003csub\u003e600\u003c/sub\u003e of 0.38, while the induction was conducted with 1 mM IPTG at an OD\u003csub\u003e600\u003c/sub\u003e of 0.77.\u003c/p\u003e \u003cp\u003eFractionally labeled \u003csup\u003e13\u003c/sup\u003eC(10%)-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab was obtained by expressing the protein in a mixture of 10% \u003csup\u003e13\u003c/sup\u003eC6 glucose and 90% un-labeled glucose as the sole carbon source.\u003c/p\u003e \u003cp\u003eProtein purifications were conducted using a fast dilution approach at pH 9.0 and with 2 M L-arginine, as described in previously (Gagn\u0026eacute; et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). His tag was removed by incubating the protein with 100 units/mg of thrombin in phosphate buffer at pH 7.0 for 5h at 37℃ under light agitation. Thrombin and Histag were removed with a Hitrap SP (5 mL), followed by size exclusion chromatography using HiLoad 26/60 Superdex 75 pg. Protein yield (before cleavage) was 37, 44, and 36 mg/L of culture were obtained for \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab, \u003csup\u003e1\u003c/sup\u003eH-I(δ1)LVmethyl-\u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab, and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC(10%)-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab, respectively. Sample for resonance assignment contained 395 \u0026micro;M \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab (21 mg/mL) in 20 mM sodium acetate-d\u003csub\u003e3\u003c/sub\u003e at pH 5.0 with 5% v/v deuterium oxide for lock frequency purposes in 50 \u0026micro;L and transferred in a 1.7 mm tube. Sample for side-chain methyl groups, prepared as described above with the addition of labelled intermediates as described by Goto and coworkers (Goto et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), contained 400 \u0026micro;M \u003csup\u003e1\u003c/sup\u003eH-I(δ1)LVmethyl-\u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab in 300 \u0026micro;L (5 mm Shigemi tube) in the same buffer. Data were collected at 40℃ (313 K) on Bruker AVANCE NEO 600 MHz (side-chain assignment), AVANCE III-HD 700 MHz (backbone assignment) and AVANCE NEO 1GHz NMR spectrometers equipped with 5mm, 1.7mm, and 5mm, respectively, TCI cryogenically cooled triple resonance inverse probeheads fitted with z-axis gradients. Chemical shift resonances were referenced with sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNMR experiments\u003c/h2\u003e \u003cp\u003eData collection for the assignment of the backbone resonances used the TROSY-based version(Eletsky et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) of the standard pulse sequences with deuterium decoupling during carbon evolution from the Bruker library: 2D-\u003csup\u003e15\u003c/sup\u003eNHSQC (trosyetf3gpsi), 3D-HNCO (trhncogp2h3d) (Salzmann et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), 3D-HN(CA)CO(Clubb and Wagner, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), 3D-HNCA (trhncagp2h3d2), 3D-HN(CO)CA (trhncocagp2h3d) (Eletsky et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), 3D-HNCACB (trhncacbgp2h3d), 3D-HN(CO)CACB (trhncocacbgp2h3d) (Grzesiek and Bax, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Eletsky et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) on the 700 MHz NMR spectrometer fitted with the 1.7 mm NMR probehead. All proton-nitrogen planes were collected using a spectral width (SW) of 18 ppm with 2048 real points (\u003csup\u003e1\u003c/sup\u003eH) and 40 ppm with 64 real points (\u003csup\u003e15\u003c/sup\u003eN). The \u003csup\u003e13\u003c/sup\u003eC indirect dimensions were collected with a SW of 14 ppm, and 128 real points for HNCO/HNCACO, a SW of 30 ppm with 128 real points for HNCA/NHCOCA, and a SW of 80 ppm with 128 real points for HNCACB/HNCOCACB. The acquisition time of 13 h (46 h) for the HNCO (TROSY) and 52 h for all other 3D experiments was used.\u003c/p\u003e \u003cp\u003eSide-chain methyl resonance of isoleucines (delta), leucines and valines were assigned with pulse sequences based on a carbon TOCSY element to transfer the methyl carbon magnetization down to either the alpha carbon or carbonyl prior to transfer to the bonded nitrogen and then proton for detection. These experiments are most efficient at fields of 600 MHz or less. Data were collected on a sample of \u003csup\u003e1\u003c/sup\u003eH-I(δ1)LVmethyl-\u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab at 465 \u0026micro;M (25 mg/mL) in 20 mM sodium acetate-d3 at pH 5.0 with 5% v/v deuterium oxide, in a 5 mm Shigemi tube. NMR pulse sequence codes were graciously provided by Prof. Lewis Kay (University of Toronto). In total a series of four 3D experiments 3D-CCC(CO)NH, 3D-HCC(CO)NH, 3D-CCC(CA)NH, and 3D-HCC(CA)NH were acquired (Tugarinov and Kay, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Data were collected with a SW of 16 ppm with 2048 real points in the proton direct dimension and a SW of 40 ppm with 64 real points in the nitrogen dimension centered at 120 ppm. The indirect proton dimensions (HCC-) were collected with a SW of 3 ppm with 64 real points and the indirect carbon dimensions (CCC-) with a SW of 22 ppm with 54 real points for a total acquisition time of 54 h and 64 h, respectively. Stereoassignment of the Pro-R and Pro-S of methyl groups of valine and leucine side chains was carried out following the method of Neri et al (Neri et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Briefly, a constant time 2D-\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC CT-HSQC was recorded on a \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC(10%)-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab sample using a constant time of 28 ms during carbon evolution. The resulting spectrum provided resonances of opposite phases for the Pro-R and Pro-S methyl groups (Tugarinov and Kay, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis and resonance assignment and validation\u003c/h2\u003e \u003cp\u003eNMR data were processed using nmrPipe (Delaglio et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) that was run via the NMRBox web facility (Maciejewski et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and visualized with NMRViewJ (Johnson and Blevins, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Semi-automated sequential assignment was carried out with the Runabout tool of NMRViewJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eValidation of NMR assignment\u003c/h2\u003e \u003cp\u003eThe web server of I-PINE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://i-pine.nmrfam.wisc.edu/index.html\u003c/span\u003e\u003cspan address=\"http://i-pine.nmrfam.wisc.edu/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Lee et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) was used to verify and validate the trastuzumab-scFab assignments. All peak lists from TROSY-based NMR experiments, namely 2D-\u003csup\u003e15\u003c/sup\u003eN-HSQC, 3D-HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB, were used as input files supplemented with the Runabout-manual assignment list and the three-dimensional X-ray structure (PDB ID 5xhg). The output of the server allowed the identification and correction of a few assignments, the identification of new assignments. In addition, we tested the new assignment protocol BARASA (Bishop et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) running under NMRBox using the same peak list and assignments as input data used for I-PINE. A total of 20 runs were conducted, using 80 concurrent threads with 0.99 convergence p-value, and a stepwise energy drop of -100. Ca, Cb, and CO zero points were all set to 0.20, with a chemical shift energy range of -50 to 100.\u003c/p\u003e \u003cp\u003eSecondary structure predictions based on using \u003csup\u003e13\u003c/sup\u003eCa, \u003csup\u003e13\u003c/sup\u003eCb, \u003csup\u003e13\u003c/sup\u003eCO, and \u003csup\u003e15\u003c/sup\u003eN chemical shift resonances of \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN-trastuzumab-scFab were performed using the web server of CIS 3.0 (Hafsa et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://csi3.wishartlab.com/cgi-bin/index.php\u003c/span\u003e\u003cspan address=\"http://csi3.wishartlab.com/cgi-bin/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TALOS-N (Bartels et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) inside NMRBox (Maciejewski et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Secondary structure elements predicted from chemical shifts were compared to the X-ray structure (PDB ID 5xhg). The validated assignments were deposited in the BioMagResBank under accession number 52228.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cem\u003eResonance assignment of backbone atoms\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eAttempts to remove the linker after thrombin cleavage of the fused poly-histidine tag yielded significant loss of sample. \u0026nbsp;Therefore, we compared the 2D-\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC of trastuzumab-scFab with the fully cleaved trastuzumab-Fab. \u0026nbsp;The extra resonances belonging to the linker (scFab) were well resolved from any backbone resonances of the Fab and all backbone resonances of both samples were overlapped, indicating that the assignment of the scFab can be directly used for the Fab (\u003cstrong\u003eFig 1\u003c/strong\u003e). \u0026nbsp; The fragment contained a total of 485 amino acids (50.8 kDa) with the heavy having 230 residues (13 prolines) and the light having 214 residues (12 prolines). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e2\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e15\u003c/sup\u003eN two-dimensional TROSY HSQC spectrum of trastuzumab-scFab shows well-dispersed resonances, typical of well-folded Fab (\u003cstrong\u003eFig 2\u003c/strong\u003e). \u0026nbsp;A total of 405 (96.7%) \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN backbone peaks were assigned, with 204 (94.0%) and 201 (99.5%) in the heavy and light chains, respectively. \u0026nbsp;Assigned carbons include 416 (93.7%) \u003csup\u003e13\u003c/sup\u003eCO, 422 (95.0%) \u003csup\u003e13\u003c/sup\u003eCa, and 373 (91.0%) non-glycine \u003csup\u003e13\u003c/sup\u003eCb. \u0026nbsp;From the 41 residues containing linker, the first two glycines were assigned and the last seven residues including the thrombin site. \u0026nbsp;The Fab fragment is composed of four immunoglobulin domains that are each stabilized by one disulfide bond: Cys24-Cys98 (heavy chain, VH), Cys149-Cys205 (heavy chain, CH1), Cys294-Cys359 (light chain, VL), Cys405-Cys465 (light chain, CL), and one bond that links the heavy to the light chain Cys225-Cys485. \u0026nbsp;All cysteine Cb chemical shifts are higher than 35 ppm, which is indicative of properly formed disulfide bonds, while reduced cysteine would have chemical shifts less than 35 ppm (Schulte\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 2020). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eResonance assignment of isoleucine delta-1, leucine and valine methyl groups\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eUsing the carbon TOCSY versions of experiments, a total of 11 isoleucines (100%), 28 leucines (93%), and 35 valines (92%) were assigned (\u003cstrong\u003eFig 3\u003c/strong\u003e). \u0026nbsp;Only four residues have not been assigned, namely Leu20, Leu198, Val93, Val193.\u003c/p\u003e\n\u003cp\u003eAnalysis of the 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC constant time HMQC experiment on a 10% \u003csup\u003e13\u003c/sup\u003eC-labeled (90% natural abundance) sample, provided complete stereoassignment for all 28 leucine and 35 valine methyl groups. \u0026nbsp;We used the 1983 IUPAC-IUC recommendation for the identification of the stereospecificity of methyl groups, with Pro-R and Pro-S being identified as g1 or d1, and g2 or d2 for valine and leucine, respectively (Markley\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 1998). \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eComparison with NISTmAb assignment\u003c/h2\u003e\n\u003cp\u003eTrastuzumab and the NIST-mAb are two monoclonal antibodies of the IgG1 class with light chain kappa. \u0026nbsp;They both share identical primary sequences in their constant heavy 1 (C\u003csub\u003eH\u003c/sub\u003e1) and constant light domain (C\u003csub\u003eL\u003c/sub\u003e). \u0026nbsp; In order to further validate our assignment, we compared it to the resonance assignment of backbone atoms of the NISTmAb-Fab(Solomon\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 2023). \u0026nbsp;It is expected that resonances arising from amides with the same local magnetic environment will have the same chemical shifts while others that have similar or slightly different environments will produce slightly or significantly different chemical shifts. \u0026nbsp; Indeed, comparison of assigned amide groups with same or very similar chemical shifts from both mAbs yielded the same assignment.\u003c/p\u003e\n\u003ch2\u003eValidation of the backbone assignment\u003c/h2\u003e\n\u003cp\u003eI-PINE was used to validate the current assignment and to help in identifying non-assigned residues. \u0026nbsp;From a total of 419 non-proline residues, I-PINE assigned 403 (96.21%) residues thus providing 15 new assignments. \u0026nbsp;From this total, 370 (95.4%) matched our assignment. \u0026nbsp;The Linear Analysis of Chemical Shifts (LACS) identified 2 outliers: Ser19 in \u003csup\u003e13\u003c/sup\u003eC-O, and Val95 in \u003csup\u003e13\u003c/sup\u003eCa and \u003csup\u003e13\u003c/sup\u003eCb. \u0026nbsp;All cysteines are fully oxidized, with the exception of Cys98, with 74.9% oxidation. \u0026nbsp;Prolines\u0026apos; isomerization state is mostly trans, with the exception of Pro158, Pro279, and Pro412. \u0026nbsp;Predominantly trans prolines are associated with folded proteins (Alderson\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInitial attempts using BARASA using default parameters to validate the assignment led to poor results: \u0026nbsp;only 195 (46.5%) residues out of the 419 were identified (Table 1). \u0026nbsp;However, optimization of the parameters increased the number of assigned residues to 361 (86.2%), corresponding to 77.4% and 95.5% of the residues of the heavy and light chains, respectively. \u0026nbsp; \u0026nbsp;While BARASA did not provide a higher number of assigned residues, the approach did allow the identification of errors or glitches in the semi-automatic assignment performed with RunAbout such as misinterpretation of which resonance belonged to the Cb(i) vs Cb( i-1) etc. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1: Parameters optimization in BARASA. \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eStepwise energy drop\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e-100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e-500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e-1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e-2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003eConvergence p-value: 0.99; Min. chemical shift energy: -50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eNo. of assignments\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e351\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e328\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eMatching assignments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e335 (92.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e326 (92.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e312 (93.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e309 (94.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eConvergence p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003eStepwise energy drop: -2000; Min. chemical shift energy: -50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eNo. of assignments\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eMatching assignments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e187 (95.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e309 (94.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eMin chem. shift energy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e-100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003eStepwise energy drop: -2000; Convergence p-value: 0.99\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eNo. of assignments\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.24%\" valign=\"top\"\u003e\n \u003cp\u003eMatching assignments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e309 (94.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e318 (92.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.44%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Default parameters: stepwise energy drop: -2000; convergence p-value: 0.99; Min. chemical shift energy: -50\u003c/p\u003e\n\u003cp\u003e1. The number of assignments corresponds to the results returned from the calculations\u003c/p\u003e\n\u003cp\u003e2. Matching assignment between BMRB 52228 and the results returned from BARASA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to test the ability of BARASA to perform \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003eresonance assignment of trastuzumab-Fab, we used a SHIFTX2 predicted list of backbone chemical shifts and a mix of \u0026lsquo;known\u0026rsquo; and predicted chemical shifts. \u0026nbsp; The list of known chemical shifts were built from residues of the constant domains C\u003csub\u003eH\u003c/sub\u003e1 and C\u003csub\u003eL\u003c/sub\u003e of trastuzumab-Fab and NIST-Fab that shares the same chemical shifts. \u0026nbsp;All remaining (unknown) residues were predicted with SHIFTX2. \u0026nbsp;Finally, a test with SHIFTX2 predicted chemical shifts was carried out. \u0026nbsp;The results (Table 2) showed that both approaches produced very good assignments (over 88%) with a very good assignment of the light chain (\u0026gt;90%). \u0026nbsp;Both procedures obtained lower assignments on the heavy chain, similar to the manual-semi-automatic assignment. \u0026nbsp;This region of the Fab domain showed lower spectral resolution and was more challenging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2: \u0026nbsp; Test of \u003cem\u003ede novo\u003c/em\u003e assignments using BARASA using the above the following parameters: convergence p-value: 0.99; Stepwise energy drop: -100; Min. chemical shift energy: -50\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"618\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003ePre-assign List\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003eRunAbout\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003eMIX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003eSHIFTX2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003eNo. of assignments\u003csup\u003e1\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003e370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e335\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003eMatching assignments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e335/361 (92.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003e328/370 (88.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e304/335 (90.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003eMatching HC-Variable (117)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e73/81 (90.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003e68/93 (73.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e67/83 (80.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003eMatching HC-Constant (100)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e80/87 (92.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003e78/87 (89.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e66/69 (95.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003eMatching LC-Variable (102)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e90/94 (95.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003e88/92 (95.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e86/94 (91.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.89320388349515%\" valign=\"top\"\u003e\n \u003cp\u003eMatching LC-Constant (101)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e92/99 (92.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.388349514563107%\" valign=\"top\"\u003e\n \u003cp\u003e94/98 (95.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.359223300970875%\" valign=\"top\"\u003e\n \u003cp\u003e85/89 (95.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes: total number of non-proline residues (excluding the linker) =420 \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1. The number of assignments corresponds to the results returned from the calculations\u003c/p\u003e\n\u003cp\u003e2. Matching assignment between BMRB 52228 and the results returned from BARASA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Matching assignment between BMRB 52228 and corresponding domains: V\u003csub\u003eH\u003c/sub\u003e, C\u003csub\u003eH\u003c/sub\u003e1, V\u003csub\u003eL\u003c/sub\u003e, and C\u003csub\u003eL\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrediction of secondary structure elements using CIS3.0 and TALOS-N allowed further validation of the assignments. \u0026nbsp;Predicted values of backbone torsion angles, visualized as secondary structure elements, are consistent with the X-ray structure (PDB ID 5xhg) (\u003cstrong\u003eFig 4\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003emAb: monoclonal antibody.\u003c/p\u003e\n\u003cp\u003escFab: single-chain fragment antigen-binding.\u003c/p\u003e\n\u003cp\u003eFab: fragment antigen-binding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVH: heavy chain variable domain.\u003c/p\u003e\n\u003cp\u003eVL: light chain variable domain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemical shifts and Bruker raw data \u003cem\u003eser\u003c/em\u003e files were deposited in the BMRB data bank with entry number 52228.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Yves Aubin; Investigation: Donald Gagn\u0026eacute;, James Aramini; Formal Analysis: Donald Gagn\u0026eacute;, James Aramini, Visualization: Donald Gagn\u0026eacute;, Yves Aubin; Writing-original draft: Donald Gagn\u0026eacute;, Yves Aubin; Writing-review and editing: Donald Gagn\u0026eacute;, Yves Aubin; Supervision: Yves Aubin.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Michael Johnston and Huixin Lu for critical reading of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eALDERSON, T. R., LEE, J. H., CHARLIER, C., YING, J. \u0026amp; BAX, A. 2018. Propensity for cis-Proline Formation in Unfolded Proteins. \u003cem\u003eChembiochem,\u003c/em\u003e 19\u003cstrong\u003e,\u003c/strong\u003e 37-42. DOI: 10.1002/cbic.201700548.\u003c/li\u003e\n \u003cli\u003eBARTELS, C., BILLETER, M., GUNTERT, P. \u0026amp; WUTHRICH, K. 1996. Automated sequence-specific NMR assignment of homologous proteins using the program GARANT. \u003cem\u003eJ Biomol NMR,\u003c/em\u003e 7\u003cstrong\u003e,\u003c/strong\u003e 207-13. DOI: 10.1007/BF00202037.\u003c/li\u003e\n \u003cli\u003eBISHOP, A. C., TORRES-MONTALVO, G., KOTARU, S., MIMUN, K. \u0026amp; WAND, A. J. 2023. 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Herceptin-induced Inhibition of Phosphatidylinositol-3 Kinase and Akt Is Required for Antibody-mediated Effects on p27, Cyclin D1, and Antitumor Action1. \u003cem\u003eCancer Research,\u003c/em\u003e 62\u003cstrong\u003e,\u003c/strong\u003e 4132-4141.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biomolecular-nmr-assignments","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnmr","sideBox":"Learn more about [Biomolecular NMR Assignments](http://link.springer.com/journal/12104)","snPcode":"12104","submissionUrl":"https://submission.nature.com/new-submission/12104/3","title":"Biomolecular NMR Assignments","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monoclonal antibody, trastuzumab, NMR spectroscopy, fragment antigen binding, Escherichia coli","lastPublishedDoi":"10.21203/rs.3.rs-4165568/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4165568/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTrastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 11:20:01","doi":"10.21203/rs.3.rs-4165568/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-19T15:04:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-19T15:03:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"05ff4072-5b75-454a-ad89-456b80152c61","date":"2024-04-01T09:09:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-01T09:07:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-26T10:11:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-26T10:11:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biomolecular NMR Assignments","date":"2024-03-25T20:45:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biomolecular-nmr-assignments","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnmr","sideBox":"Learn more about [Biomolecular NMR Assignments](http://link.springer.com/journal/12104)","snPcode":"12104","submissionUrl":"https://submission.nature.com/new-submission/12104/3","title":"Biomolecular NMR Assignments","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"24aa4cce-3885-4e4f-941f-543ff5373917","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T03:58:56+00:00","versionOfRecord":{"articleIdentity":"rs-4165568","link":"https://doi.org/10.1007/s12104-024-10177-3","journal":{"identity":"biomolecular-nmr-assignments","isVorOnly":false,"title":"Biomolecular NMR Assignments"},"publishedOn":"2024-05-08 03:58:55","publishedOnDateReadable":"May 8th, 2024"},"versionCreatedAt":"2024-03-29 11:20:01","video":"","vorDoi":"10.1007/s12104-024-10177-3","vorDoiUrl":"https://doi.org/10.1007/s12104-024-10177-3","workflowStages":[]},"version":"v1","identity":"rs-4165568","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4165568","identity":"rs-4165568","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}