Cross-linked protein crystals with an intense nonconventional full-color photoluminescence originating from through-space intermolecular interaction

Cross-linked protein crystals with an intense nonconventional full-color photoluminescence originating from through-space intermolecular interaction | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Aggregate This is a preprint and has not been peer reviewed. Data may be preliminary. 10 April 2025 V1 Latest version Share on Cross-linked protein crystals with an intense nonconventional full-color photoluminescence originating from through-space intermolecular interaction Authors : Renbin Zhou , Xiaoli Lu , Xuefeng Zhou , Xuejiao Liu , Shanmin Wang , Tymish Ohulchanskyy 0000-0002-7051-6534 , Da-Chuan Yin , and Junle Qu 0000-0001-7833-4711 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174425063.37058260/v1 Published Aggregate Version of record Peer review timeline 350 views 186 downloads Contents Abstract Introduction Results 2.2.2. Two-photon excited photoluminescence of lysozyme crystals 2.3 Assessing structure-emission relationship 2.4 Rational color tuning of lysozyme crystal emission Conclusions Experimental methods Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The emergence of nonconventional luminescent materials (NLMs) has attracted significant attention due to their sustainable synthesis and tunable optical properties. Yet, establishing a clear structure-emission relationship remains a challenge. In this work, we report a previously unknown class of NLMs: cross-linked protein crystals that exhibit intense photoluminescence (PL) in the visible range (425–680 nm). We systematically investigated seven natural protein crystals (concanavalin, catalase, lysozyme, hemoglobin, α-chymotrypsin, pepsin, and β-lactoglobulin) cross-linked with glutaraldehyde and demonstrated that cross-linking induces broadband emission that is absent in natural crystals. Focusing on polymorphic lysozyme crystals (tetragonal, orthorhombic, monoclinic), we found excitation-dependent fluorescence with lifetimes in the nanosecond range and quantum yields up to 20% (in the monoclinic phase under 450 nm excitation). Single- and two-photon spectroscopy, as well as pressure- and solvent-modulated PL studies, confirm that the emission is due to intermolecular through-space interactions (TSI) within the crystal lattice. Compression enhances TSI and red-shifts the emission, whereas the solvent (DMSO) induced swelling reduces TSI and causes a blue shift, establishing a direct structure-emission correlation. This work establishes protein crystals as programmable NLMs with tunable emission and provides a mechanistic framework for the design of nonconventional luminogens through protein crystal engineering. Introduction Nonconventional luminescent materials (NLMs) lacking aromatic luminophores have emerged as a reserch hotspot due to their facile manufacture, cost-effectiveness and environmental sustainablility [1, 2] . NLMs are typically characterized by electron-rich heteroatoms with lone-pair electrons (N, O, S, and P) or unsaturated bonds (C═O, C═C, C═N, and C≡N), previously unrecognized as emission centers [3, 4] . The clustering-triggered emission (CTE) is proposed to explain the unconventional emission behavior of NLMs [5] . While non-conjugated heteroatoms or unsaturated bonds exhibit negligible fluorescence in diluted solutions, “clusters” of electron-rich heteroatoms and/or unsaturated bonds in molecular aggregate state enable electron clouds partly overlapped, resulting in prolonged through-space interaction (TSI) by inter-/intramolecular n–π, π–π, and/or n–n interactions [6, 7] . Moreover, the substantial TSI rigidifies the conformation, resulting in inhibition of the nonradiative decay and, therefore greatly promoting radiative deactivation of an electronic excitation (i.e., fluorescence emission) [8-10] . CTE theory is frequently utilized to explain the emission characteristics of NLMs, there are still many issues that remain unsolved and require further studies. In particular, the following questions arise: which heteroatoms and/or unsaturated bonds are involved in the formation of nonconventional luminophores in NLMs and what are the requirements to their spatial distribution. [11] To address these queries, it is necessary to clearly determine the nature and structure of nonconventional luminophores [12] . Due to the fact that the majority of NLMs have been accidentally discovered and derived from various natural compounds (such as cellulose [13] , monosaccharides, rosin [14] ), or synthetic polymers (such as polyetheramines, polyborate [15] , etc.), it is difficult to employ a systematic, (e.g., single crystal based) approach to determine common structural details of nonconventional luminophores [16, 17] . For a comprehensive elucidation of the nonconventional luminescence mechanism, more NLMs with extensive structural information (particularly in the solid state) must be considered. It should be noted that a continual adjustment of emission color to achieve full-color emission is important for practical applications [17-19] . Despite their excitation-dependent emission behavior, the majority of NLMs emit mostly in the blue spectral region [20] . In order to obtain an efficient emission at longer wavelengths (particularly, in the red and near-infrared ranges), a few pioneering works introduced an approach, where the same structural design as for conventional luminous materials was considered [21] . For instance, bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride (DA) has been modified by an introduction of C═C bridging bonds, which resulted in a redshift of the emission from blue to yellowish-white [22] . Modification of cyclodextrin (CD) with non-florescent amino acids enabled the generation of blue to cyan fluorescence [23] . Anionic polymerization of poly(maleimide)s was shown to achieve a full-color emission [24] . Due to the unknown structure of nonconventional luminophores and their aggregates, direct regulation of the NLMs emission color is considered to be problematic [23, 25] . It is highly desirable to construct and explore NLMs with defined structure in the aggregate (solid) state to rationally control the luminescence emission color. Protein crystals can specifically meet the abovementioned criterion of the defined structure in solid state and provide a basis for a development of new NLMs. In particular, protein crystals, formed when protein molecules assemble into a highly ordered structure via non-covalent interactions are rich in heteroatoms and unsaturated bonds, which is beneficial for formation of NLMs [26, 27] . As a result, bovine serum albumin (BSA) protein in powder and several amino acid crystals have been reported to manifest unconventional emission [28, 29] . It should be noted that the variable structure of the protein crystals (polymorphism) is always associated with varied optical physical properties, allowing for the establishment of a clear structure-emission relationship. Furthermore, polymorphic protein crystals can be readily prepared [30] and X-ray single diffraction can determine the crystal structure with atomic resolution. Herein, we report cross-linked protein crystals as a new class of NLMs that exhibit excitation-dependent full-color emission with packing-dependent characteristics, a phenomenon that has never been described before. The micro- or millimeter-sized crystals of seven randomly picked proteins (i.e., concanavalin, catalase, lysozyme, hemoglobin, α-chymotrypsin, pepsin and β-lactoglobulin) were first synthesized. As the protein crystals are known to be, in general, unstable, a cross-linking with glutaraldehyde has been employed to improve stability of the crystals [31] . All the synthesized cross-linked crystals revealed unconventional full-color photoluminescence (PL) emission, which was brightly visible in all emission channels of the fluorescence microscope. Next, polymorphic lysozyme crystals (with tetragonal, orthorhombic and monoclinic structures, Figure 1a) were selected for a thorough study to explore PL characteristics and establish an unambiguous structure-emission relationship. The PL of the lysozyme crystals was found to cover whole visible range (~400-700 nm), be excitation dependent, and had lifetimes in a range of nanoseconds, which allowed us to term it fluorescence. Single- and two-photon excited fluorescence excitation and emission spectra of the polymorphic lysozyme crystals were acquired and the absolute fluorescence yields were measured. Study of the photophysical and structural properties of cross-linked lysozyme crystals revealed that the PL emission was originated from the intermolecular TSI; a stronger intermolecular TSI is associated with the emission at longer wavelengths (Figure 1b). Consequently, the PL color was shown to be tunable by manipulating the intermolecular TSI in lysozyme crystals. In particular, the redshift of PL emission was achieved by means of high pressure applied to lysozyme crystals, which led to the crystal compressing and a corresponding increase in intermolecular TSI. To the contrary, when lysozyme crystals were swollen in organic solvent (DMSO), a blue-shift in PL emission was observed, which could be associated with a decrease in an intermolecular TSI (Figure 1c). This work not only introduces cross-linked protein crystals as a new class of full-color NLMs, but also provides a reliable strategy to reveal the structural details of nonconventional luminophores examining the structure-emission relationship and rationally tuning the nonconventional PL emission to the desired spectral range. Figure 1. Scheme illustrating a structure-emission relationship in polymorphic lysozyme crystals and emission color tuning through crystal swelling or compressing. Results 2.1 Full-color emission of protein crystals revealed by fluorescence microscopy It is well known that protein molecular solutions display intrinsic PL in ultraviolet (300-350 nm) spectral range due to the aromatic luminophores of tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) [32] . With the exception of fluorescent proteins, which emit due to the availability of the specific fluorophore fixed within the protective β-barrel [33] , a phenomenon of visible PL emission from non-fluorescent proteins in visible range is sparsely mentioned. The majority of studies have exclusively focused on the UV-emitting protein molecules in solutions [28] , while the visible photoluminescence of proteins in solid state has attracted little attention. We have accidentally discovered that catalase and hemoglobin protein nanocrystals, which were synthesized and employed by us for drug delivery in cancer therapy [34-36] , emit in broad visible range and their full-color emission can be visualized using epifluorescence or confocal laser scanning microscopy (CLSM). The unexpected and unusual visible PL emission of these protein nanocrystals has intrigued us and motivated for further studies. First, number of natural protein (concanavalin, catalase, lysozyme, hemoglobin, α-chymotryps, pepsin and β-lactoglobulin) crystals with sizes in micro- or millimeter scale were prepared using the protein crystallization method [36, 37] , with the crystallization parameters specified in Table S1 in Supporting Information. Next, CLSM was employed to check an existence of their multicolor emission. As shown in Figure S1 (Supporting Information), all the tested protein crystals exhibited unconventional full-color emission observable in all four fluorescence channels of the confocal laser scanning microscope (λ ex =405nm, λ em =425-475 nm; λ ex =488nm, λ em =500-550nm; λ ex =561nm, λ em =570-620 nm; λ ex =640 nm, λ em =660-680nm). In contrast, control CLSM of NaCl crystals did not show any signal in the fluorescence channels of the confocal microscope, indicating that the observable full-color emission is not an artefact but it does originate from the synthesized protein crystals. It should be noted that protein crystals are known for their fragility and instability, which limits their applications. At the same time, their stability and mechanical properties can be enhanced through cross-linking [38] . In this regard, an established method of cross-linking with glutaraldehyde used in our previous works on protein nanocrystals [34-36] has been applied by us to the micro- or millimeter scale crystals synthesized in this study. On the other hand, cross-linking has been reported to enhance the PL of carbon dots, carbonized polymer dots and polymers [39-41] , suggesting that PL in protein crystals can be enhanced as a result of cross-linking. Figure S2 shows that both cross-linked and non-cross-linked lysozyme crystals display PL emission. However, the PL of cross-linked crystals is found to be hugely more intense than that of the non-cross-linked ones (Figure S3). Therefore, the cross-linked crystals were further characterized and considered as a novel class of NLMs with color-tunable PL emission. 2.2. Luminescence spectroscopy characterization 2.2.1. Single-photon excited PL emission of lysozyme crystals To investigate photophysical characteristics of cross-linked protein crystals and the underlying mechanism of the observed emission, lysozyme crystals were chosen due to the ease of their crystallization with polymorphic structures [42] . By altering the crystallization conditions (Table S2), three types of submillimeter lysozyme crystals with distinct morphologies (tetragonal, orthorhombic and monoclinic) were produced and their PL emission and excitation spectra were recorded (Figure 2). As shown in Figure 2a, a molecular solution of lysozyme (0.1 mg/mL) only emits at 300-350 nm, when excited at 280-300 nm; this emission is obviously the intrinsic fluorescence of protein molecules. In contrast, the synthesized lysozyme crystals of all three morphologies emitted fluorescence in a broad spectral range of 280 nm-650 nm (Figure 2c, e, g). Notably, under excitation at peaked at ~340 nm, which matches the fluorescence of lysozyme molecules and can be assigned to the aromatic residues (Trp, Tyr and Phe) [28, 43] . As shown in Figure 2b, a peak at 280 nm dominates in the absorption spectra of both the molecular solution and the crystals, revealing the characteristic absorption of protein molecules. At the same time, no clear absorption peaks could be seen at longer wavelengths suggests that additional non-identified absorption centers appear after crystallization. Figure 2. Absorption and PL of cross-linked lysozyme crystals. (a): PL emission spectra of lysozyme molecular solution (0.1 mg/mL in water) excited at different wavelengths; (b) normalized UV-VIS absorption spectra of lysozyme molecular solution and lysozyme crystals of different morphology; (c, e, g): fluorescence emission spectra of polymorphic lysozyme crystals and corresponding transmission and fluorescence microscopy images of the crystals; (d, f, h) CIE chromaticity coordinates of polymorphic lysozyme crystals calculated from their emission spectra at varying λ ex . Further spectroscopy studies have revealed that the obtained lysozyme crystals exhibit emission with excitation-dependent behavior. In particular, when the excitation wavelength λ ex is tuned from 280 nm to 550 nm, the PL emission wavelengths gradually shift toward longer wavelengths in deep red spectral range (Figure 2c). Moreover, the PL emission of the tetragonal lysozyme crystals under an intense 532 nm laser irradiation has been shown to extend even to near infrared (NIR) spectral region, reaching beyond 800 nm (Figure S4), which is rarely observed in other NLMs. It should be also noted that under the excitation at longer wavelengths (310nm-550nm), all three types of lysozyme crystals manifest a significantly more intense emission than under excitation with shorter wavelengths (280-300nm), suggesting that the new emission centers formed after crystallization are more efficient than the aromatic residues. It is worth mentioning that such an excitation-dependent and spectrally broad PL is very similar to that manifested by nanoscale carbon particles (carbon dots, CDs) upon surface passivation. The excitation-dependent emission from CDs is associated with a broad set of luminescence centers of specific energy levels that are photo-selected by the excitation wavelength [44] . The CDs are now emerging as major multicolor luminescent nanomaterials in biomedical applications and mechanisms of their multicolor PL are believed to be associated with many factors, such as size, surface states, element doping, hydrogen bonds, surface functionalization, energy transfer, cross-linked enhanced emission (CEE) effect, etc. [45] . Similarly, mechanisms of the multicolor PL manifested by protein crystals can be complex. To further assess the multicolor feature of the emission from lysozyme crystals, the CIE (Commission International de I’Eclairage) chromaticity coordinate diagrams were calculated for lysozyme crystals of all three morphologies, revealing the full color blue-green-red emission (Figures 2d, 2f, 2h). As it is discussed above, the excitation-dependent full-color emission of the cross-linked lysozyme crystals can be associated by the formation of a variety of new emission centers, similarly to the case of CDs. In the excitation spectra for lysozyme in molecular form, only the 280 nm emission peak is observed (Figure S5a), however for lysozyme crystals (Figure S5b-5d), with emission wavelengths λ em of 300-700 nm, several excitation peaks are observed, confirming the presence of several types of emission centers in the lysozyme crystals. Figure 3. Time-resolved PL spectroscopy of lysozyme crystals and their photostability under two-photon excitation. (a-c): PL decays for tetragonal (a), orthorhombic (b) and monoclinic (c) lysozyme crystals dispersed in water. (d): Two-photon induced (840 nm laser irradiation) photobleaching of tetragonal lysozyme crystals in water dispersion in comparison with that for rhodamine 6G aqueous solution. To further investigate the emission centers coexisting in lysozyme crystals, the PL emission decays were recorded. As shown in Figure 3a, 3b and 3c, the decays of the emission from lysozyme crystals are in nanosecond range and can be fitted using a double-exponential model; the nanosecond scale indicates that the emission can be attributed to a fluorescence (rather than to a long-lived phosphorescence). With the use of different excitation lasers (370nm, 455nm and 570nm) to excite fluorescence of the lysozyme crystals, the lifetime varies, once again revealing the existence of multiple emission centers in the crystals. This was also supported by the absolute quantum yield measurements. An excitation of the same crystals with varied excitation wavelengths (350nm, 400nm, 450nm, 500nm, and 550nm) led to different quantum yields (Table S3). Importantly, the highest fluorescence quantum yield was determined to be almost 20% (19.75% for monoclinic crystals). As cross-linked lysozyme crystals exhibit abnormal full-color fluorescence (apparently due to a creation of multiple emission centers during crystallization) with significant emission yield, they can be considered as NMLs. 2.2.2. Two-photon excited photoluminescence of lysozyme crystals To assess a possibility of two-photon excitation for the protein crystal fluorescence, the two-photon excited fluorescence (TPEF) spectra of polymorphic lysozyme crystals were acquired using excitation of a femtosecond laser tunable in 760 nm - 1100 nm range. As showed in Figure S6, under this excitation, lysozyme crystals of all three morphologies exhibited excitation-dependent emission, the emission color varies from blue to green to red, exhibiting the same full-color emission behavior as in case of the single photon excitation. Moreover, the two-photon fluorescence lifetime changes with excitation wavelength and crystals type (Figure S7-S10), demonstrating that the multiple emission clusters coexist in the crystals and determine the full-color emission. It is worth noting that the photostability of the lysosome crystal fluorophores under two-photon excitation is significantly better than that for molecular fluorophores that we assessed (i.e., rhodamine 6G). Figure 3d demonstrates that after 840 nm two photon laser irradiation (10 mW/cm 2 ) for 10 min, tetragonal lysozyme crystal retained about 75% of the initial fluorescence intensity, while fluorescence of rhodamine 6G aqueous solution completely disappeared after four minutes of irradiation, suggesting that photostability of lysozyme crystals could be sufficient for future applications. Based on the one-photon and two-photon photophysical properties, distinctive full-color emission behavior is achieved in all three polymorphisms of lysozyme crystals as well as other protein crystals. This is a highly desirable but challenging characteristic of NLMs. The color-tunable emission is connected with the formation of various emissive species. Moreover, all three polymorphism lysozyme crystals exhibited similar excitation-dependent full-color emission; however, different crystalline types exhibited dissimilar fluorescence properties (maximum emission wavelength, lifetime and absolute quantum yield), providing an ideal model for establishing a precisely structure−emission relationship to elucidate the underlying mechanism. 2.3 Assessing structure-emission relationship Generally, it is considered to be quite challenging to explain the mechanism of NLMs emission due to their undefined structure [46] . Currently, the CTE is reported to explain emission mechanism for NLMs, [47] and the CTE theory can explain their unique full-color emission of protein crystals. However, the CTE mechanism is far from being a universally satisfactory mechanism for protein crystal emission. As follows from the results presented in Figures 2, 3, S5-S10, and Table S3, different packing modes for the same lysozyme molecules result in different photophysical properties of the crystals (emission wavelengths, quantum yield and lifetime). It appears to be insufficient to explain the distinct difference only by the restriction of the molecular motion as a result of crystal rigidification; consequently, with more structural details on a formation of unconventional luminophore clusters are required for a deeper understanding of the underlying mechanism. The atomic structural details of crystals can be determined by X-ray diffraction crystallography, which provides a reliable platform for establishing a clear structure-properties relationship to uncover the underlying mechanism of protein crystal emission. We determined the structure of lysozyme crystals of different morphology using X-ray single crystallography. The structural details of the polymorphic lysozyme crystal of three structures are presented in Table S4; the results are consistent with previously reported studies [48-50] . Alignment of the three lysozyme structures with different morphologies showed complete overlap (RMSD value = 0.01, Figure S11), indicating identical intramolecular interactions and excluding their influence on emission. Thus, the only difference between the polymorphisms lies in their intermolecular interactions, which may contribute to the original emission of lysozyme crystals. The X-ray crystallography studies show that lysozyme crystals have the monoclinic P12 1 1 space group and orthorhombic P2 1 2 1 2 1 space group, with four lysozyme molecules per unit cell. Along with that, lysozyme crystals have a tetragonal P4 3 2 1 2 space group with eight lysozyme molecules per unit cell (Figure 4a-c). Taking one unit cell as an example, as depicted in Figure S12, multiple emission centers are indeed created by TSI between neighboring molecules in each of the three types of lysozyme crystals. The electron-rich heteroatoms N and O cluster together by many conventional N-H···O=C hydrogen bonds, and the distances and spatial locations of the heteroatoms can be clearly identified based on structural details (Figure 4a-c). In addition, abundant short contacts between neighboring molecules, such as H-C···O=C, N-H···C=O, N-H···C-H, H-N···N-H, and H-N···O=C interactions, would establish a strong TSI network to expand the electron delocalization for the emission (Figure S13-S15). The structure analysis also reveals that the distance between hydrogen bonds (Figure 4d) and multiple short contacts (Figure S16) is less than 3.0 Å, whereas the Van-der-Waals Radius atomic radius of C, N and O is 1.7 Å, 1.55 Å and 1.52 Å, respectively, indicating the strong interaction and electron communication among C, N and O atoms, which results in more efficient TSIs and increases the emission ability of clusters. Obviously, these hydrogen bonds and short contacts significantly restrict molecular movements, rigidify the crystal structure, and increase emission. To study further the effect of intermolecular interaction on emission properties, the intermolecular interaction strength and distance were systematically calculated for three types of lysozyme crystals. Tetragonal lysozyme crystal has the greatest number of hydrogen bonds and shortest hydrogen bonds (2.2-2.7 Å), according to the obtained results (the half number of hydrogen bonds was considered because of the double lysozyme molecules in the unit cell). The number of hydrogen bonds in the monoclinic lysozyme crystal was found to be the lowest of the three types (Figure 4d). At the same time, as one can see in Figure 4e, the emission spectra (under the same excitation wavelength) are somewhat more red-shifted for tetragonal crystals than for orthorhombic crystals than for monoclinic crystals, suggesting that the stronger intermolecular TSI do result in a red shift of the emission. It is worth noting that the TPEF peak depends similarly on the excitation wavelength (Figure S17). Consequently, our findings allow to discriminate experimentally the formed emission centers in lysozyme crystals. In addition, quantitative data indicated that intermolecular interactions play a vital role in tunable full-color emission of the lysozyme crystals, with stronger interactions resulting in longer wavelength emission. Figure 4. Structure-emission relationship for lysozyme crystals. (a-c) structural details of polymorphic lysozyme crystals; (d) intermolecular hydrogen bonds of polymorphic lysozyme crystals; (e) dependence of the spectral position of the polymorphic lysozyme crystals emission peak on excitation wavelength. 2.4 Rational color tuning of lysozyme crystal emission A rational control of the emission properties of NLMs remains a challenge. Traditional techniques for controlling the emission of NLMs involved mostly chemical covalent changes of precursor monomer molecules to alter their electronic energy levels. Rarely the aggregate state of NLMs can be directly and rationally regulated to alter emission behavior. Considering the importance of the intermolecular TSIs to the emission of lysozyme crystals, the emission behavior could be dynamically manipulated by changing the intermolecular TSIs. According to the structure-emission relationship, increase in the intermolecular TSIs in lysozyme crystal would result in a red shift of the fluorescence emission, while decrease in the intermolecular TSIs produces a blue shift. The intermolecular TSIs in tetragonal lysozyme crystals was decreased by the crystal swelling due to an interaction with the organic solvent DMSO (Figure 5a). After screening, crystal swelling was indistinguishable at concentrations of DMSO below 60%, however, a substantial crystal volume increase was found at concentration of DMSO more than 60% (70%, 80% and 90%). As depicted in Figure S18, the crystal volume of a tetragonal lysozyme crystal treated with 70% DMSO doubled within 60 min. When the concentration of DMSO was increased to 80%, it was evident that crystal expansion had begun from the crystal outside to its core, and the maximum swelling had occurred within 20 min (Figure 5c). When the DMSO concentration is increased to 90%, the expansion rate of crystals is significantly accelerated, with the maximum expansion occurring just 10 min later (Figure 5d). In addition, as shown in Figure S18, Figure 5c and Figure 5d, with the same excitation and detection parameters, the emission intensity of swollen crystals was lower that of native lysozyme crystals, and the higher the degree of swelling, the lower the corresponding fluorescence intensity in all the SLCM channels. Furthermore, the emission spectra of tetragonal lysozyme crystals were measured prior to and after swelling. As expected, as the degree of crystal swelling increased, the emission intensity of crystals dropped, and the maximum emission peak moved blue (Figure 5b, Figure S19). Experiments confirmed that weakening the intermolecular TSI of a tetragonal lysozyme crystal by swelling will result in emission attenuation and wavelength blue shift. In addition, a compact monoclinic lysozyme crystal to strengthen the intermolecular TSI was created by applying high pressure with a diamond anvil cell (DAC) (Figure 5e). As shown in Figure 5f and 5g, the emission behavior of lysozyme crystal can be broken down into three stages. At the first stage, when the pressure changes from 0 GPa to 1.05 GPa, the emission intensity of the lysozyme crystal increases gradually, and a new emission peak at about 600 nm is emerges alongside the red shift of the maximum emission wavelength. In the second stage, as the pressure is increased from 1.05 GPa to 2.88 GPa, the emission intensity of the lysozyme crystal is gradually diminished, but the emission wavelengths do not significantly change, though some red shift (if to compare with spectra at the first stage). At the third stage, when gradually reducing the pressure from 2.88 GPa to 0 GPa, the emission intensity is gradually increasing, and the emission spectra appear to be even more red shifted, a shoulder at peak around 650 nm appeared. Figure 5. Rational tuning of the lysozyme crystal emission color (a) swelling tetragonal lysozyme crystal with DMSO; (b) corresponding emission spectra treated with 80% and 90% DMSO under 800 nm two-photon laser irradiation; (c) optical and fluorescence images of tetragonal lysozyme crystals treated with 80% DMSO for 20min; (d) optical and fluorescence images of tetragonal lysozyme crystals treated with 90% DMSO for 10min; (e) compacting the monoclinic lysozyme crystal with a diamond anvil cell; (f) corresponding emission spectra of monoclinic lysozyme crystal under 532 nm one photon laser irradiation under different pressure; (g) in situ optical images of monoclinic lysozyme crystal compressed by different pressures; (h) Schematic illustrations of the mechanism for multiple emissions of lysozyme crystal under swelling and compacting. The following explanations apply to this phenomenon: at stage one, the loaded pressure (0 GPa to 1.05 GPa) is weaker, the structure of the lysozyme crystal remains unchanged (Figure 5f), and the adjacent lysozyme molecules in the crystal are forced to approach one another, further enhancing the TSIs and rigidifying the conformation, causing the energy gap to decrease, the emission intensity to increase, and emission peak to redshift. Which is supported by the in situ absorption spectrum. As expected, the in situ UV-Vis absorption spectra gradually changed to blue, indicating the band gap of the lysozyme crystal narrowed as the loading pressure increased (Figure S20). At stage two, the crystal appeared to undergo a transformation or transition to a different crystal phase or amorphous phase, resulting in the destruction of the generated emission cluster and a considerable change in the intermolecular TSIs, which decreases the emission (Figure 5f). At stage three, when the loaded pressure is released, the shape of the lysozyme crystal partially recovers, new emission centers may emerge, and the emission intensity and emission peak may shift. Consequently, the crystal swelling and compressing results demonstrated that the emission of lysozyme crystal can be controlled by adjusting the intermolecular TSIs directly in the solid state. Increasing the intermolecular TSI will redshift the emission, while decreasing it will result in blue-shifted emission. These findings demonstrate the intermolecular TSI contribution to lysozyme crystal the emission is reliable. According to abovementioned findings, the tunable full-color emission of lysozyme crystals could be illustrated schematically (Figure 5h). In dilute lysozyme dilute solutions, no TSIs clusters are formed, and only the intrinsic UV emission originated from aromatic residues can be observed. In contrast, during crystallization, intermolecular interaction between neighboring molecules in the crystal form multiple emission clusters with multiple vibrational energy levels through TSIs. Upon excitation from the ground state (S 0 ) to the excited state (S 1 ), the excitons release energy through the radiative channels of TSIs, exhibiting an obvious excitation-dependent emission behavior. It is worth noting that there is an interesting correlation between the strength of TSIs and the emission wavelengths: an increase in TSIs strength leads to a red-shifting of the emission peak. By adjusting the intermolecular TSIs, the emission color of lysozyme crystals could therefore be successfully tuned. When the intermolecular TSIs was weakened by expanding the crystal with DMSO, the energy gap widened and a blue-shifted emission was observed. Conversely, the intermolecular TSIs increases as a result of the crystal compressing under high pressure, electron delocalization is extended and the energy gap reduces, resulting in a red shift of the emission. Conclusions In this work, cross-linked protein crystals are introduced as a new type of NLMs with excitation dependent and spectrally tunable full-color fluorescence. Optical spectroscopy (SPEF and TPEF emission spectra, excitation spectra, fluorescence lifetimes, and fluorescence quantum yields) demonstrated that there are various new emission centers formed after protein crystallization, and the cross-linking stabilizes the formed emission centers for enhanced emission. Benefiting the atomic resolution structure details of polymorphic lysozyme crystals (tetragonal, orthorhombic and monoclinic structure) with their photophysical properties, an unambiguous structure-emission relationship reveals that intermolecular TSIs between adjacent molecules is essential for achieving full-color emission, and stronger intermolecular TSIs results in red-shifted emission. Consequently, the emission color of lysozyme crystals is adjusted by modifying the intermolecular TSIs. Compressing the crystal at high pressure to increase TSIs efficiency results in longer-wavelength emission. While weakening the TSI by swelling the crystal with DMSO leads to shorter wavelength emission, providing a straightforward and direct method for regulating the emission color of NLMs by external stimulus (solvent and pressure). This work not only reported a novel class of NLMs, but also established a structure-emission relationship for the in-depth elucidation of emission mechanisms. In addition, this work provides a new and general strategy for directly manipulating the “aggregate” intermolecular TSIs to fine-tune the emission color, so endowing them with high practical utility. Experimental methods 4.1 Materials and reagents Lysozyme lyophilized powder (L4919, ≥40, 000 units/mg), catalase lyophilized powder (C40, ≥10, 000 units/mg), concanavalin A lyophilized powder (C5275), pepsin lyophilized powder (P6887, ≥3, 200 units/mg), β-lactoglobulin lyophilized powder (L3908, ≥90%), α-chymotrypsin lyophilized powder (C4129, ≥40 units/mg) were purchased from Sigma-Aldrich (St. Louis, USA) without further purification. Hemoglobin was purified from chicken blood by protein crystallization methods. Protein crystallization screen kits were obtained from Hampton research (California, USA). Sodium chloride, lithium nitrate, acetic acid, sodium acetate and glutaraldehyde were supplied by Servicebio (Beijing, China). 4.2 Preparation of protein crystals All the protein crystals were prepared by batch crystallization methods. Catalase crystals were grown from a crystallization solution containing 8 mg/mL catalase with the crystallization reagent (0.1 M MES, 20% PEG 3350, pH 6.3, 20 ℃). Hemoglobin crystals were grown from a crystallization solution containing 50 mg/mL hemoglobin with the crystallization reagent (1× PBS, 25% PEG3350, pH7.4, 20 ℃). Tetragonal lysozyme crystals were grown from a crystallization solution containing 40 mg/mL lysozyme with the crystallization reagent (0.1 M sodium acetate, 1.4 M NaCl, pH 4.6, 20 ℃). Tetragonal lysozyme crystals were grown from a crystallization solution containing 20 mg/mL lysozyme with the crystallization reagent (0.1 M sodium acetate, 0.3 M NaCl, pH 4.6, 20 ℃); Orthorhombic lysozyme crystals were grown from a crystallization solution containing 40 mg/mL lysozyme with the crystallization reagent (0.1 M sodium acetate, 1.4 M NaCl, pH 4.6, 40 ℃); α-chymotrypsin, pepsin and β-lactoglobulin protein crystals were imaged with the lyophilized powder. After obtaining the crystals, they were cross-linked by 1% glutaraldehyde to improve the stability for further studies [31] . 4.3 Characterization of lysozyme crystals The absorption spectra of lysozyme crystals were acquired using a UV/VIS/NIR spectrophotometer LAMBDA 750 (PerkinElmer). The fluorescence spectra and lifetime of lysozyme crystals were measured using a spectrofluorometer Fluorolog-3 equipped with a FluoroCube system (Horiba). During fluorescence lifetime measurements, three excitation sources from Horiba (NanoLEDs emitting at 370nm，455 nm and 570 nm) were employed; the fluorescence decays were acquired at the wavelengths of fluorescence maxima. The absolute fluorescence quantum yield was determined using an integrating sphere (Horiba Quanta–φ). The fluorescence microscopy images of protein crystals were acquired using confocal laser scanning microscope Nikon A1. All the four available excitation lasers (405 nm, 488 nm, 561 nm and 640 nm) were used to excite and image the fluorescence of the same crystals. The two photon excited fluorescence microscopy images were obtained with confocal laser scanning microscope Nikon A1 equipped with a femtosecond pulsed laser tunable from 760nm to 1300 nm (Chameleon Discovery from Coherent). The same laser and an Ocean Optics spectrometer were used to obtain the two-photon excited fluorescence emission spectra. The two-photon excited fluorescence decays were collected using TCSPC systems (SPC-150 and DCC-100, Becker & Hickl GmbH) and a Chameleon Discoverylaser as an excitation source. 4.4 Determination of lysozyme crystals structure To determine the structure of lysozyme crystals, they were frozen in liquid nitrogen with the cryoprotectant of 5% glycerol in reservoir solution. The diffraction data of lysozyme crystals were collected at Synchrotron Radiation Facility (Shanghai, China). The collected data were processed by HKL2000 software, and the final lysozyme structures were refined by CCP4 and COOT. 4.5 Structural analysis of lysozyme crystals The unit cells of three lysozyme crystal structures were generated using Chimera (https://www.cgl.ucsf.edu/chimera/). MD Analysis 1 and in-house scripts were used to analyze the number of contacts and hydrogen bonds between the monomers in each unit cell. Intermolecular contacts were counted with a distance cutoff range of 2.2 Å to 3.6 Å. Hydrogen bonds were defined with a donor-acceptor distance cutoff range of 2.2 Å to 3.6 Å, and a donor-hydrogen-acceptor angle cutoff of 150°. To visually demonstrate the intermolecular interactions, structural images were generated using PyMOL [51] . 4.6 Compressing and swelling of lysozyme crystals structure Compressing of lysozyme crystals. Orthorhombic lysozyme crystal was compressed with a diamond anvil cell (DAC, IIa). A diamond with 500 μm diameter culet was used to generate high pressure. A 250 μm thickness T301 stainless steel gasket was prepared to a thickness of 50 μm and drilled a central hole of 200μm to load the orthorhombic lysozyme crystal. Water was used to transmit the pressure. The loaded pressure was in situ calibrated by Ruby spheres together inserted into the central hole. The fluorescence spectra of orthorhombic lysozyme crystals under varying pressure at room temperature were measured in situ using a confocal Raman microscope (Horiba LabRAM HR Evolution), excitation wavelength: 532nm, scan range: 500 nm-850 nm. The absorption spectra of orthorhombic lysozyme crystal under various pressure were in situ measured using a UV–Vis absorption spectrophotometer (Ocean Optics DH-2000-BAL). The wavelengths ranged from 250 to 700 nm. Swelling of lysozyme crystals. Single tetragonal lysozyme crystal was transferred from water solution into the microscopy Petri dish using a Cryo-Loop (Hampton research). Then 1mL of 20 vol% DMSO solution was added into the dish and it was incubated for 5h. The same tetragonal lysozyme crystal was serially transferred into increasing DMSO solution (30 vol%, 40 vol%, 50 vol%, 60 vol%) for 5h. For final three higher DMSO solution (70 vol%, 80 vol%, 90 vol%), the crystal was swelling quickly, and the corresponding fluorescent images and fluorescence spectra were captured timely. 4.7 Microscopy imaging of cells treated with lysozyme nanocrystals 4T1 cells (murine mammary carcinoma cell line) were seeded in 35 mm Petri dishes at the cell density of 1 × 10 4 . After incubation for 24 hours, cells were treated with tetragonal lysozyme crystals (5 μM) for 4h. After rinsing and addition of the fresh cell medium, the cells were imaged in all four fluorescence channels of the confocal laser scanning microscope confocal (λ ex = 405 nm, λ em = 425 –475 nm; λ ex = 488 nm, λ em = 500–550 nm; λ ex = 561 nm, λ em = 570 –620 nm). Two-photon excited fluorescence imaging were also performed using confocal microscope equipped with a Chameleon Ti:sapphire laser tuned to 840 nm. TPEF lifetime was acquired using time-correlated single-photon counting (TCSPC) system. TPEF was excited at 840 nm and fluorescence decays were collected and fitted with double-exponential decay functions. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors acknowledge support from the National Key R&D Program of China (2021YFF0502900); National Natural Science Foundation of China (62127819, T2421003, 62435011, 32471230, W2431056); Shenzhen Key Laboratory of Photonics and Biophotonics (ZDSYS20210623092006020); Shenzhen Science and Technology Program (JCYJ20220818100202005), and Fundamental Research Funds for the Central Universities (23GH02021). References [1] Tang, Saixing, Tianjia Yang, Zihao Zhao, et al., Chemical Society Reviews. 2021 . 50(22),12616-12655. [2] Zhang, Haoke, Zheng Zhao, Paul R McGonigal, et al., Materials Today. 2020 . 32,275-292. 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Information & Authors Information Version history V1 Version 1 10 April 2025 Peer review timeline Published Aggregate Version of Record 3 Jun 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Aggregate Keywords intermolecular through-space interactions nonconventional luminescent materials polymorphism protein crystals structure-emission relationship Authors Affiliations Renbin Zhou Northwestern Polytechnical University School of Life Sciences View all articles by this author Xiaoli Lu Westlake University School of Life Sciences View all articles by this author Xuefeng Zhou Southern University of Science and Technology Department of Electrical and Electronic Engineering View all articles by this author Xuejiao Liu Northwestern Polytechnical University School of Life Sciences View all articles by this author Shanmin Wang Southern University of Science and Technology Department of Electrical and Electronic Engineering View all articles by this author Tymish Ohulchanskyy 0000-0002-7051-6534 Shenzhen University School of Physics and Optoelectronic Engineering View all articles by this author Da-Chuan Yin Northwestern Polytechnical University School of Life Sciences View all articles by this author Junle Qu 0000-0001-7833-4711 [email protected] Shenzhen University School of Physics and Optoelectronic Engineering View all articles by this author Metrics & Citations Metrics Article Usage 350 views 186 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Renbin Zhou, Xiaoli Lu, Xuefeng Zhou, et al. Cross-linked protein crystals with an intense nonconventional full-color photoluminescence originating from through-space intermolecular interaction. Authorea . 10 April 2025. DOI: https://doi.org/10.22541/au.174425063.37058260/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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