H, 15 N and 13 C resonance assignments of S2A and H64A double mutant of human carbonic anhydrase II

H, 15 N and 13 C resonance assignments of S2A and H64A double mutant of human carbonic anhydrase II | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article H, 15 N and 13 C resonance assignments of S2A and H64A double mutant of human carbonic anhydrase II Neelam #, Mandar Bopardikar, Himanshu Singh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4565827/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Sep, 2024 Read the published version in Biomolecular NMR Assignments → Version 1 posted 7 You are reading this latest preprint version Abstract Protein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause increase in hydrophobicity in N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn 2+ -binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for 1 H, 15 N, and 13 C. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn 2+ -binding pocket and its role in catalysis. NMR resonance assignments Ser2Ala His64Ala hCAII Figures Figure 1 Figure 2 Figure 3 Biological context Human carbonic anhydrase II (hCAII) is an enzyme of significant physiological importance, belonging to the carbonic anhydrase family. These enzymes catalyze the rapid conversion of carbon dioxide and water to bicarbonate (HCO₃⁻) and protons (H⁺), a reversible reaction that is crucial for various biological processes (Krishnamurthy et al. 2008 ). hCAII is one of the most studied and fastest enzymes in the carbonic anhydrase family, showcasing high efficiency in catalyzing this reaction (Lindskog 1997 ). hCAII is a zinc metalloenzyme, with a zinc ion in its active site that is essential for its catalytic activity (Kiefer and Fierke 1994 ). The enzyme consists of a single polypeptide chain and has a molecular weight of about 29 kDa. The zinc ion is coordinated by three histidine residues (Kiefer and Fierke 1994 ) and a hydroxide ion (Lipton et al. 2004 ) that acts as a nucleophile in the catalysis process (Lipton et al. 2004 ). The primary function of hCAII, and carbonic anhydrases in general, is to maintain acid-base balance in blood and other tissues (Occhipinti and Boron 2019 ). The bicarbonate and protons rapidly produced from CO₂ by hCAII can then be easily transported in the bloodstream. This process is crucial in the kidneys for the reabsorption of bicarbonate and in the lungs for the release of CO₂ during respiration. hCAII also plays a role in the formation of aqueous humor in the eye (Wistrand et. al. 1986 ) and protecting oesophageal, gastric, and intestinal mucosa from acidity (Parkkila et. al. 1994 ; Lönnerholm et. al. 1985 ). Mutations in the gene encoding hCAII can lead to diseases such as osteopetrosis (Sly et. al. 1983 ; Shah et. al. 2004 ), renal tubular acidosis (Nagai et. al. 1997 ), and cerebral calcification (Sly et. al. 1983 , 1985 ). Inhibitors of carbonic anhydrase, such as acetazolamide (Sippel et. al. 2009 ; Vidgren et. al. 1990 ), are used clinically to treat conditions like glaucoma, epilepsy, altitude sickness, and certain types of edema, by reducing the production of bicarbonate. Due to its role in pH regulation and CO₂ transport, hCAII and its inhibitors have been studied for potential applications in managing conditions related to carbon dioxide and bicarbonate transport, as well as for carbon capture and sequestration efforts to mitigate climate change impacts (Effendi and Ng 2019 ). Recent research has shown that change in hCAII levels could be a factor in Alzheimer's Disease (Jang et. al. 2010 ). Also, mitochondria associated hCAII has been identified as a potential target for modulating neurodegeneration and age-related impairments (Pollard 2016). Our project delves into the intricate role of enzyme dynamics in catalysis, a topic that has sparked considerable debate over the years. There has been a wealth of research focusing on how enzymes' structure and dynamics are affected by their interaction with water, a key factor in biomolecular processes. Although X-ray crystallography has proven effective in identifying structural waters, capturing the dynamic interplay within the water-enzyme network has been challenging. In our previous research, we utilized solid-state NMR relaxation dispersion analysis to investigate the dynamics at the active site of hCAII (Vasa et al. 2018 , 2019 ; Singh et al. 2020 ), which is crucial for the catalytic action of carbonic anhydrases. This approach, together with chemical shift information, relaxation analysis, and molecular dynamics simulations, revealed significant microsecond-scale dynamics throughout the enzyme's water network, as detailed in our findings (Singh et al. 2019 ). Interestingly, introducing an inhibitor to this system slowed down these dynamics and disturbed the network to some extent (Vasa et al. 2019 ). The dwell time of zinc-bound water in the enzyme's pocket, as inferred from earlier magnetic relaxation dispersion studies (Denisov et al. 1999 ), matched the timing of these dynamic movements, underscoring their importance in catalytic efficiency (Vasa et al. 2018 , 2019 ; Singh et al. 2020 ). Current study explores the restructuring of the enzyme's water pocket structure and its consequences for the catalytic function of hCAII. We are specifically focused on the conformational changes that occur during the critical proton transport process in hCAII, examining if the movement of excess protons correlates with conformational changes in the protein and the water molecules in the Zn 2+ -pocket, as suggested by Taraphder et al. ( 2016 ). The mechanism by which enzymes promote proton transfer via dynamic networks of hydrogen bonds between amino acid residues and water molecules is well-documented (Nagle and Morowitz, 1978 ; Nagle and Tristram-Nagle 1983 ). The critical function of His64 in facilitating proton movement, acting mainly as a donor or acceptor, is highlighted by the significant decrease in proton transfer when His64 is replaced with alanine (Tu et al. 1989 ). Alterations in Trp5, Tyr7, Asn62, and Gln92 have also been observed to impact the enzyme's catalytic performance (Tu et al. 2002 ; Elder et al. 2004 ; Paul and Taraphder 2015 ). Neutron imaging suggests that a network of hydrogen-bonded water molecules within hCAII's active site links the zinc-associated water to the internal configuration of His64 (Roy and Taraphder, 2007 ; Maupin and Voth 2010 ). Studies using molecular dynamics have shown that when His64 faces inward, a chain of 3–4 water molecules is sufficient for effective proton transfer, whereas networks of 5–6 water molecules can facilitate even greater proton movement (Roy and Taraphder 2006 ; Maupin et al. 2008 ; Maupin and Voth 2010 ; Mikulski and Silverman 2010 ). The configuration of these water molecule clusters and the positioning of His64 are crucial for the enzyme's catalytic effectiveness (An et al. 2002 ; Maupin and Voth 2010 ). On the other hand, the Ser2 residue is thought to be involved in the entry of water into the Zn 2+ -binding pocket. Therefore, in view of this significant role of H64 in the function of hCAII and that of S2 as a gate-keeper for water, we commence by detailing the near-complete sequence-specific resonance assignments for the Ser2Ala/His64Ala variant of hCAII. This foundational work is poised to pave the way for subsequent explorations into the complex nature and arrangement of the water-protein network within hCAII. Essentially, our study aims to shed light on the elaborate connectivity of water molecules within the enzyme's active site, revealing the dynamic interactions that drive its catalytic efficiency. Methods and experiments Protein expression and purification The cDNA for the S2A/H64A double mutant was effectively cloned and overexpressed in BL21 (DE3) E. coli . This protein was then purified to absolute purity using a protocol outlined in prior studies (Vasa et al. 2018 ; Singh et al. 2019 , 2020 ). Initially, a pGEX expression vector, which included the S2A mutant hCAII gene, was utilized. To introduce the His64Ala mutation, site-directed mutagenesis was applied. The accuracy of the hCAII gene sequence was thoroughly verified by DNA sequencing, performed meticulously with SnapGene software. A single transformed colony was selected and grown in E. coli culture under specific conditions: in M9 medium enriched with 1g/L 15N-ammonium chloride and 2g/L 13C-glucose, maintained at 37°C. The growth induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was carefully timed, initiating when the culture's optical density at 600 nm (OD 600 ) reached between 0.6 and 0.8, to ensure optimal protein yield. Following its overexpression, the protein's purification followed the previously published protocols (Singh et al. 2019 , 2020 ), leading to the isolation of the S2A/H64A mutant protein in its purest form. NMR spectroscopy Protein samples were prepared using a solvent consisting of 90% water (H2O) and 10% heavy water (D2O), along with a 100 mM phosphate buffer at pH 6.4, containing 0.3 mM of the protein. These samples were analyzed using a Bruker Avance 700 MHz NMR spectrometer equipped with a 5 mm triple-resonance cryogenic probe. The analysis featured sensitivity-enhanced 2D [ 15 N- 1 H]-HSQC experiment as described by Vallurupalli et al. ( 2012 ), which included a water-flipback technique to minimize water saturation effects, and a range of 3D NMR techniques namely CBCA(CO)NH (Grzesiek and Bax 1993 ; Muhandiram and Kay 1994 ), HNCA (Schleucher et al. 1993 ) and HNCO (Schleucher et al. 1993 ; Kay et al. 1994 ). The HSQC spectrum was acquired with 2048 and 256 complex points along 1 H and 15 N dimensions, respectively. These experiments were performed at a temperature of 310 K on uniformly 15 N/ 13 C-labeled S2A/H64A double mutant of hCAII, primarily to assign backbone resonances. Additionally, [ 15 N- 1 H]-NOESY-HSQC (with a 100 ms mixing time) was conducted as per Muhandiram et al. ( 1993 ). The NMR data were processed using Bruker Topspin 3.1 software and analyzed using CARA software (Keeler, 2010 ). Chemical shifts for 15 N and 13 C were calibrated indirectly, whereas 1 H chemical shifts were directly referenced to the external standard, 2,2-dimethyl-2-silapentene-5-sulfonate (DSS), according to Edison et al. ( 1994 ) and Chary and Govil ( 2008 ). Extent of assignment and data deposition Sequence-specific resonance assignments of the S2A/H64A double mutant of hCAII were nearly complete for 1 H, 13 C, and 15 N. We successfully assigned 93% of the backbone 1 H/ 15 N, 75% CO, and 93% Cα peaks. The 1 H N and 15 N assignments are displayed in the 2D [ 15 N- 1 H]-HSQC (Fig. 1 ). In the 3D-HNCO spectrum, we anticipated 244 resonances from non-proline residues, and under the given experimental conditions, we observed 235 peaks (96% of the expected), all of which were clearly identified. Additionally, 56% of sidechain aliphatic 1 H resonances were assigned, whereas, 92% of sidechain aliphatic 13 C resonance assignments were obtained, Identification of Gln and Asn residues was facilitated by their sidechain 1 H/ 15 N resonances. The chemical shift data have been deposited in the BioMagResBank ( http://www.bmrb.wisc.edu ) under accession number 52498. Missing correlations were noted for residues H4, H10, N11, F20, H119, P200, and A243. Interestingly, in the S2A mutant of hCAII, correlations were missing for residues A2, H3, N11, T199, P200, and P201 (Neelam and Singh 2024 ). This variation in the absence of correlations may indicate structural or dynamic differences between the S2A and S2A/H64A mutants. The secondary structural propensity plot for the S2A/H64A double mutant of hCAII, derived from the differences in chemical shift values of 13 CO, 13 Cα, 13 Cβ, 1 H N , and 15 N relative to random coil values (Wishart and Sykes 1994 ) using the Neighbor Corrected Structural Propensity Calculator ( https://st-protein02.chem.au.dk/ncSPC/ ), predicted five α helices and ten β strands, aligning with the NMR structure of hCAII (pdb id: 3ks3). These predictions were further confirmed by NOEs from 15 N-edited NOESY-HSQC. Comparing the 2D 15 N− 1 H HSQC spectra of the S2A/H64A double mutant and wild-type hCAII, recorded under similar experimental conditions, revealed chemical shift perturbations for the W16 sidechain, L57, the N62 sidechain, G63, N67 backbone and sidechain, and R245 (Fig. 3 ), suggesting structural differences between the mutant and the wild-type protein. Additionally, no peak broadening was observed for the L84, N62, N67 sidechains, and R245 in the S2A/H64A double mutant compared to wild-type hCAII. R245, N62, and N67 sidechains are part of the conserved protein-water network in the wild-type enzyme and thus experience fast chemical exchange between water-bound and unbound forms. The absence of broadening for these resonances in the HSQC spectrum of the S2A/H64A double mutant may indicate a reduced exchange rate of water within the active-site pocket. These assignments lay the groundwork for a more detailed investigation into the role of the water network in the function of hCAII. Further studies on the dynamics of water exchange and its relation to protein function are ongoing. Declarations Acknowledgements Technical support from the high field NMR at IISER Berhampur is acknowledged. Author contributions H.S., M.B. and N. designed and conducted experiments, analyzed data, prepared figures, and wrote the manuscript. M.B. and H.S. also deposited assignments in the BMRB. Data availability Assignments of S2A/H64A hCAII has been deposited in the BMRB under accession code 52498. Conflict of interest No competing interests Ethical Approval Not applicable. Funding The Ramalingaswami re-entry fellowship, Department of Biotechnology, New Delhi, India. Consent for publication Each author gives their approval for it to be published. Availability of data and materials Data could be shared upon request. References An H, Tu C, Duda D, et al (2002) Chemical rescue in catalysis by human carbonic anhydrases II and III. 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Cite Share Download PDF Status: Published Journal Publication published 21 Sep, 2024 Read the published version in Biomolecular NMR Assignments → Version 1 posted Editorial decision: Revision requested 14 Aug, 2024 Reviews received at journal 14 Aug, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers invited by journal 14 Jun, 2024 Editor assigned by journal 11 Jun, 2024 Submission checks completed at journal 11 Jun, 2024 First submitted to journal 11 Jun, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4565827","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317646260,"identity":"b945c4bb-a05e-4544-9390-4eccc602774e","order_by":0,"name":"Neelam #","email":"","orcid":"","institution":"Indian Institute of Science Education and Research Berhampur","correspondingAuthor":false,"prefix":"","firstName":"Neelam","middleName":"","lastName":"#","suffix":""},{"id":317646261,"identity":"9afd7150-f3cc-4887-9326-c6293e355784","order_by":1,"name":"Mandar Bopardikar","email":"","orcid":"","institution":"Indian Institute of Science Education and Research Berhampur","correspondingAuthor":false,"prefix":"","firstName":"Mandar","middleName":"","lastName":"Bopardikar","suffix":""},{"id":317646262,"identity":"2e23863d-5993-4f45-a0b5-9451c129f6f0","order_by":2,"name":"Himanshu Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDACZhCqkOCBciWI1XJGgoeHeC0gXYxtDAw8hNVBgXw77+HXhfMsZOz5FzB++MFgkUdQi8FhvjTrmduADpN4wCzZwyBRTFgLM4+ZMS9YywEGaaBfEhsIOqwZpGUOWAvzb6K0MBzmMX7M2wDUwt/ARpwtBod5zJh5jgG13GBss+wxIMZh/WeMP/PU1Nmz9x8+fONHRR0RDmNgYINEH9h8AyLUAwHzBzDFf4A45aNgFIyCUTDyAACtzi3BLvwSUwAAAABJRU5ErkJggg==","orcid":"","institution":"Indian Institute of Technology Guwahati","correspondingAuthor":true,"prefix":"","firstName":"Himanshu","middleName":"","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2024-06-11 17:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4565827/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4565827/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12104-024-10203-4","type":"published","date":"2024-09-21T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59964436,"identity":"cbe06505-2b26-48b5-a7bd-f5d2f6afd464","added_by":"auto","created_at":"2024-07-10 01:51:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":367027,"visible":true,"origin":"","legend":"\u003cp\u003e2D [\u003csup\u003e15\u003c/sup\u003eN–\u003csup\u003e1\u003c/sup\u003eH]–HSQC of S2A and H64A double mutant of hCAII at pH 6.4 and 310 K recorded on a 700 MHz NMR spectrometer. \u0026nbsp;One letter code along with amino acid residue number along hCAII sequence indicates the respective peak assignment.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4565827/v1/b4d5f97d9429281aa25d2123.png"},{"id":59964437,"identity":"b287636a-2822-4d7d-ad60-b8851a37233a","added_by":"auto","created_at":"2024-07-10 01:51:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":229915,"visible":true,"origin":"","legend":"\u003cp\u003eThe neighbour corrected secondary structure of the S2A/H64A double mutant of hCAII, where positive and negative values are indicative of a-helical and b-sheet propensities, respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4565827/v1/8a0c2a5d35bc55428b79589a.png"},{"id":59965503,"identity":"81c062fc-2c17-4d48-b98f-7a79a0f279aa","added_by":"auto","created_at":"2024-07-10 01:59:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":282748,"visible":true,"origin":"","legend":"\u003cp\u003eAn overlay of the [\u003csup\u003e15\u003c/sup\u003eN–\u003csup\u003e1\u003c/sup\u003eH]–HSQC of wild type hCAII (black) and the S2A/H64A double mutant of the protein (red). The mutated residue A64 is shown along with the most significantly perturbed peaks.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4565827/v1/b05582a3e8dd1e4d3e5ec616.png"},{"id":65104015,"identity":"2af8938c-5ef7-4a3e-93f5-7e0cf89e827d","added_by":"auto","created_at":"2024-09-23 16:10:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1216640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4565827/v1/06a0955a-9fca-441a-acbd-0efe0a3a8ce6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"H, 15 N and 13 C resonance assignments of S2A and H64A double mutant of human carbonic anhydrase II","fulltext":[{"header":"Biological context","content":"\u003cp\u003eHuman carbonic anhydrase II (hCAII) is an enzyme of significant physiological importance, belonging to the carbonic anhydrase family. These enzymes catalyze the rapid conversion of carbon dioxide and water to bicarbonate (HCO₃⁻) and protons (H⁺), a reversible reaction that is crucial for various biological processes (Krishnamurthy et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). hCAII is one of the most studied and fastest enzymes in the carbonic anhydrase family, showcasing high efficiency in catalyzing this reaction (Lindskog \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). hCAII is a zinc metalloenzyme, with a zinc ion in its active site that is essential for its catalytic activity (Kiefer and Fierke \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The enzyme consists of a single polypeptide chain and has a molecular weight of about 29 kDa. The zinc ion is coordinated by three histidine residues (Kiefer and Fierke \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and a hydroxide ion (Lipton et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) that acts as a nucleophile in the catalysis process (Lipton et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe primary function of hCAII, and carbonic anhydrases in general, is to maintain acid-base balance in blood and other tissues (Occhipinti and Boron \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The bicarbonate and protons rapidly produced from CO₂ by hCAII can then be easily transported in the bloodstream. This process is crucial in the kidneys for the reabsorption of bicarbonate and in the lungs for the release of CO₂ during respiration. hCAII also plays a role in the formation of aqueous humor in the eye (Wistrand et. al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) and protecting oesophageal, gastric, and intestinal mucosa from acidity (Parkkila et. al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; L\u0026ouml;nnerholm et. al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMutations in the gene encoding hCAII can lead to diseases such as osteopetrosis (Sly et. al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Shah et. al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), renal tubular acidosis (Nagai et. al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), and cerebral calcification (Sly et. al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Inhibitors of carbonic anhydrase, such as acetazolamide (Sippel et. al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Vidgren et. al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), are used clinically to treat conditions like glaucoma, epilepsy, altitude sickness, and certain types of edema, by reducing the production of bicarbonate. Due to its role in pH regulation and CO₂ transport, hCAII and its inhibitors have been studied for potential applications in managing conditions related to carbon dioxide and bicarbonate transport, as well as for carbon capture and sequestration efforts to mitigate climate change impacts (Effendi and Ng \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recent research has shown that change in hCAII levels could be a factor in Alzheimer's Disease (Jang et. al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Also, mitochondria associated hCAII has been identified as a potential target for modulating neurodegeneration and age-related impairments (Pollard 2016).\u003c/p\u003e \u003cp\u003eOur project delves into the intricate role of enzyme dynamics in catalysis, a topic that has sparked considerable debate over the years. There has been a wealth of research focusing on how enzymes' structure and dynamics are affected by their interaction with water, a key factor in biomolecular processes. Although X-ray crystallography has proven effective in identifying structural waters, capturing the dynamic interplay within the water-enzyme network has been challenging. In our previous research, we utilized solid-state NMR relaxation dispersion analysis to investigate the dynamics at the active site of hCAII (Vasa et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which is crucial for the catalytic action of carbonic anhydrases. This approach, together with chemical shift information, relaxation analysis, and molecular dynamics simulations, revealed significant microsecond-scale dynamics throughout the enzyme's water network, as detailed in our findings (Singh et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Interestingly, introducing an inhibitor to this system slowed down these dynamics and disturbed the network to some extent (Vasa et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The dwell time of zinc-bound water in the enzyme's pocket, as inferred from earlier magnetic relaxation dispersion studies (Denisov et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), matched the timing of these dynamic movements, underscoring their importance in catalytic efficiency (Vasa et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrent study explores the restructuring of the enzyme's water pocket structure and its consequences for the catalytic function of hCAII. We are specifically focused on the conformational changes that occur during the critical proton transport process in hCAII, examining if the movement of excess protons correlates with conformational changes in the protein and the water molecules in the Zn\u003csup\u003e2+\u003c/sup\u003e-pocket, as suggested by Taraphder et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The mechanism by which enzymes promote proton transfer via dynamic networks of hydrogen bonds between amino acid residues and water molecules is well-documented (Nagle and Morowitz, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Nagle and Tristram-Nagle \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). The critical function of His64 in facilitating proton movement, acting mainly as a donor or acceptor, is highlighted by the significant decrease in proton transfer when His64 is replaced with alanine (Tu et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Alterations in Trp5, Tyr7, Asn62, and Gln92 have also been observed to impact the enzyme's catalytic performance (Tu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Elder et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Paul and Taraphder \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Neutron imaging suggests that a network of hydrogen-bonded water molecules within hCAII's active site links the zinc-associated water to the internal configuration of His64 (Roy and Taraphder, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Maupin and Voth \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Studies using molecular dynamics have shown that when His64 faces inward, a chain of 3\u0026ndash;4 water molecules is sufficient for effective proton transfer, whereas networks of 5\u0026ndash;6 water molecules can facilitate even greater proton movement (Roy and Taraphder \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Maupin et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Maupin and Voth \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Mikulski and Silverman \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The configuration of these water molecule clusters and the positioning of His64 are crucial for the enzyme's catalytic effectiveness (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Maupin and Voth \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). On the other hand, the Ser2 residue is thought to be involved in the entry of water into the Zn\u003csup\u003e2+\u003c/sup\u003e-binding pocket. Therefore, in view of this significant role of H64 in the function of hCAII and that of S2 as a gate-keeper for water, we commence by detailing the near-complete sequence-specific resonance assignments for the Ser2Ala/His64Ala variant of hCAII. This foundational work is poised to pave the way for subsequent explorations into the complex nature and arrangement of the water-protein network within hCAII. Essentially, our study aims to shed light on the elaborate connectivity of water molecules within the enzyme's active site, revealing the dynamic interactions that drive its catalytic efficiency.\u003c/p\u003e"},{"header":"Methods and experiments","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eThe cDNA for the S2A/H64A double mutant was effectively cloned and overexpressed in BL21 (DE3) \u003cem\u003eE. coli\u003c/em\u003e. This protein was then purified to absolute purity using a protocol outlined in prior studies (Vasa et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Initially, a pGEX expression vector, which included the S2A mutant hCAII gene, was utilized. To introduce the His64Ala mutation, site-directed mutagenesis was applied. The accuracy of the hCAII gene sequence was thoroughly verified by DNA sequencing, performed meticulously with SnapGene software. A single transformed colony was selected and grown in E. coli culture under specific conditions: in M9 medium enriched with 1g/L 15N-ammonium chloride and 2g/L 13C-glucose, maintained at 37\u0026deg;C. The growth induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was carefully timed, initiating when the culture's optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) reached between 0.6 and 0.8, to ensure optimal protein yield. Following its overexpression, the protein's purification followed the previously published protocols (Singh et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), leading to the isolation of the S2A/H64A mutant protein in its purest form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNMR spectroscopy\u003c/h2\u003e \u003cp\u003eProtein samples were prepared using a solvent consisting of 90% water (H2O) and 10% heavy water (D2O), along with a 100 mM phosphate buffer at pH 6.4, containing 0.3 mM of the protein. These samples were analyzed using a Bruker Avance 700 MHz NMR spectrometer equipped with a 5 mm triple-resonance cryogenic probe. The analysis featured sensitivity-enhanced 2D [\u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e1\u003c/sup\u003eH]-HSQC experiment as described by Vallurupalli et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which included a water-flipback technique to minimize water saturation effects, and a range of 3D NMR techniques namely CBCA(CO)NH (Grzesiek and Bax \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Muhandiram and Kay \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), HNCA (Schleucher et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and HNCO (Schleucher et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Kay et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The HSQC spectrum was acquired with 2048 and 256 complex points along \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e15\u003c/sup\u003eN dimensions, respectively. These experiments were performed at a temperature of 310 K on uniformly \u003csup\u003e15\u003c/sup\u003eN/\u003csup\u003e13\u003c/sup\u003eC-labeled S2A/H64A double mutant of hCAII, primarily to assign backbone resonances. Additionally, [\u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e1\u003c/sup\u003eH]-NOESY-HSQC (with a 100 ms mixing time) was conducted as per Muhandiram et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The NMR data were processed using Bruker Topspin 3.1 software and analyzed using CARA software (Keeler, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Chemical shifts for \u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e13\u003c/sup\u003eC were calibrated indirectly, whereas \u003csup\u003e1\u003c/sup\u003eH chemical shifts were directly referenced to the external standard, 2,2-dimethyl-2-silapentene-5-sulfonate (DSS), according to Edison et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and Chary and Govil (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtent of assignment and data deposition\u003c/h3\u003e\n\u003cp\u003eSequence-specific resonance assignments of the S2A/H64A double mutant of hCAII were nearly complete for \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, and \u003csup\u003e15\u003c/sup\u003eN. We successfully assigned 93% of the backbone \u003csup\u003e1\u003c/sup\u003eH/\u003csup\u003e15\u003c/sup\u003eN, 75% CO, and 93% Cα peaks. The \u003csup\u003e1\u003c/sup\u003eH\u003csub\u003eN\u003c/sub\u003e and \u003csup\u003e15\u003c/sup\u003eN assignments are displayed in the 2D [\u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e1\u003c/sup\u003eH]-HSQC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the 3D-HNCO spectrum, we anticipated 244 resonances from non-proline residues, and under the given experimental conditions, we observed 235 peaks (96% of the expected), all of which were clearly identified. Additionally, 56% of sidechain aliphatic \u003csup\u003e1\u003c/sup\u003eH resonances were assigned, whereas, 92% of sidechain aliphatic \u003csup\u003e13\u003c/sup\u003eC resonance assignments were obtained, Identification of Gln and Asn residues was facilitated by their sidechain \u003csup\u003e1\u003c/sup\u003eH/\u003csup\u003e15\u003c/sup\u003eN resonances. The chemical shift data have been deposited in the BioMagResBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bmrb.wisc.edu\u003c/span\u003e\u003cspan address=\"http://www.bmrb.wisc.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under accession number 52498. Missing correlations were noted for residues H4, H10, N11, F20, H119, P200, and A243. Interestingly, in the S2A mutant of hCAII, correlations were missing for residues A2, H3, N11, T199, P200, and P201 (Neelam and Singh \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This variation in the absence of correlations may indicate structural or dynamic differences between the S2A and S2A/H64A mutants. The secondary structural propensity plot for the S2A/H64A double mutant of hCAII, derived from the differences in chemical shift values of \u003csup\u003e13\u003c/sup\u003eCO, \u003csup\u003e13\u003c/sup\u003eCα, \u003csup\u003e13\u003c/sup\u003eCβ, \u003csup\u003e1\u003c/sup\u003eH\u003csub\u003eN\u003c/sub\u003e, and \u003csup\u003e15\u003c/sup\u003eN relative to random coil values (Wishart and Sykes \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) using the Neighbor Corrected Structural Propensity Calculator (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://st-protein02.chem.au.dk/ncSPC/\u003c/span\u003e\u003cspan address=\"https://st-protein02.chem.au.dk/ncSPC/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), predicted five α helices and ten β strands, aligning with the NMR structure of hCAII (pdb id: 3ks3). These predictions were further confirmed by NOEs from \u003csup\u003e15\u003c/sup\u003eN-edited NOESY-HSQC. Comparing the 2D \u003csup\u003e15\u003c/sup\u003eN\u0026minus;\u003csup\u003e1\u003c/sup\u003eH HSQC spectra of the S2A/H64A double mutant and wild-type hCAII, recorded under similar experimental conditions, revealed chemical shift perturbations for the W16 sidechain, L57, the N62 sidechain, G63, N67 backbone and sidechain, and R245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting structural differences between the mutant and the wild-type protein. Additionally, no peak broadening was observed for the L84, N62, N67 sidechains, and R245 in the S2A/H64A double mutant compared to wild-type hCAII. R245, N62, and N67 sidechains are part of the conserved protein-water network in the wild-type enzyme and thus experience fast chemical exchange between water-bound and unbound forms. The absence of broadening for these resonances in the HSQC spectrum of the S2A/H64A double mutant may indicate a reduced exchange rate of water within the active-site pocket. These assignments lay the groundwork for a more detailed investigation into the role of the water network in the function of hCAII. Further studies on the dynamics of water exchange and its relation to protein function are ongoing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eTechnical support from the high field NMR at IISER Berhampur is acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.S., M.B. and N. designed and conducted experiments, analyzed data, prepared figures, and wrote the manuscript. M.B. and H.S. also deposited assignments in the BMRB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAssignments of S2A/H64A hCAII has been deposited in the BMRB under accession code 52498.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo competing interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ramalingaswami re-entry fellowship, Department of Biotechnology, New Delhi, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEach author gives their approval for it to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData could be shared upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAn H, Tu C, Duda D, et al (2002) Chemical rescue in catalysis by human carbonic anhydrases II and III. 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Investigative Ophthalmology \u0026amp; Visual Science 27:419-428\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":"NMR resonance assignments, Ser2Ala, His64Ala, hCAII","lastPublishedDoi":"10.21203/rs.3.rs-4565827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4565827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause increase in hydrophobicity in N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn\u003csup\u003e2+\u003c/sup\u003e-binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e15\u003c/sup\u003eN, and \u003csup\u003e13\u003c/sup\u003eC. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn\u003csup\u003e2+\u003c/sup\u003e-binding pocket and its role in catalysis.\u003c/p\u003e","manuscriptTitle":"H, 15 N and 13 C resonance assignments of S2A and H64A double mutant of human carbonic anhydrase II","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-10 01:51:22","doi":"10.21203/rs.3.rs-4565827/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-14T21:50:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-14T21:49:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262798092712559215044698309958781239135","date":"2024-06-14T11:41:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-14T11:35:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-12T01:35:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-12T01:34:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biomolecular NMR Assignments","date":"2024-06-11T17:50:10+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":"66e72037-7884-460a-a283-32c4da6d9277","owner":[],"postedDate":"July 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:02:14+00:00","versionOfRecord":{"articleIdentity":"rs-4565827","link":"https://doi.org/10.1007/s12104-024-10203-4","journal":{"identity":"biomolecular-nmr-assignments","isVorOnly":false,"title":"Biomolecular NMR Assignments"},"publishedOn":"2024-09-21 15:57:29","publishedOnDateReadable":"September 21st, 2024"},"versionCreatedAt":"2024-07-10 01:51:22","video":"","vorDoi":"10.1007/s12104-024-10203-4","vorDoiUrl":"https://doi.org/10.1007/s12104-024-10203-4","workflowStages":[]},"version":"v1","identity":"rs-4565827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4565827","identity":"rs-4565827","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}