Impact of Chemical Cross-Linking on Protein Structure and Function

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Impact of Chemical Cross-Linking on Protein Structure and Function Daniel Rozbesky, Michal Rosulek, Zdenek Kukacka, Josef Chmelik, Petr Man, and Petr Novák Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02863 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Analytical Chemistry

Impact of Chemical Cross-Linking on Protein Structure and Function Daniel Rozbeský†,‡, Michal Rosůlek†,‡, Zdeněk Kukačka†,‡, Josef Chmelík†,‡, Petr Man†,‡ and Petr Novák*,†,‡ †

Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic; ‡Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic. KEYWORDS: CXMS, cross-linking, mass spectrometry, protein structure

ABSTRACT: Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information of proteins and protein complexes. Also, cross-linking has become a powerful tool for stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly it has not been investigated in detail so far. Here, we used kinetic studies, mass spectrometry, and NMR spectroscopy to systematically investigate an impact of cross-linking on structure and function of human carbonic anhydrase and alcohol dehydrogenase 1 from Saccharomyces cerevisiae. We found that cross-linking induces rather local structural disturbances and the overall fold is preserved even at a higher cross-linker concentration. The results establish general experimental conditions for chemical cross-linking with minimal effect on protein structure and function.

Introduction Chemical cross-linking coupled with mass spectrometry (CXMS) is a popular technique for structural characterization of proteins and their complexes. Since its introduction for studying protein structure by Young et al.1, CXMS has emerged as an alternative technique to traditional methods of structural biology. The great strengths of CXMS are low sample consumption, no limitation in protein size, high sensitivity, and fast analysis taking place in solution. All these aspects make CXMS a method of choice for proteins or their complexes that are inaccessible by crystallography or NMR alone. Furthermore, chemical cross-linking becomes popular for single particle cryoelectron microscopy and has been beneficial in reducing sample heterogeneity for 3D reconstruction of numerous macromolecular complexes in the past few years. In a basic approach of CXMS, a protein or protein complex is treated with a bifunctional cross-linker that can introduce a covalent bond between residues that are in close proximity to each other2. The cross-linked protein is then digested, and the resulting peptide mixture is analyzed by mass spectrometry in order to identify cross-linked peptides3. Cross-link derived constraints are ultimately used for model building4-6. The popularity of CXMS has increased steadily, and a significant progress has been made in applications, new protocols, and in improvement of bioinformatic tools in recent years7-14. The CXMS approach was successfully applied in structural analyses of various challenging macromolecular complexes such as RNA polymerase15, 16, prokaryotic ribosome17, TriC/CCT chaperonin18, 19, proteasome20-24, and membrane proteins25. Furthermore, recent advances in quantitative CXMS pave the way towards analyzing dynamics of protein conformation and protein interactions26-30. Great progress has been made in computational structural biology, and CXMS has recently

gained enough reliability to be used in combination with molecular dynamics simulations or conformational space search for de novo protein structure determination31,32. Although there is an expanding community of structural biologists utilizing chemical cross-linking, the major question for wider application remains about the possibility of distorting the protein structure and function by crosslinker. Chemical cross-linking can, in principle, trigger an artificial conformational change due to disruption in electrostatic interactions by amine or carboxyl reactive cross-linkers. Further structural perturbations may be caused by stabilizing a low-populated conformation or by limiting protein flexibility. Artificial conformational change may expose previously protected site and make it accessible to the cross-linker. Moreover, the cross-linking at high concentration of protein or cross-linker may lead to protein aggregation or an artificial oligomerization. If either of these states is subjected to further analysis, the credibility of the final result is questionable. Here we examine the effect of the most commonly used cross-linkers on the structure and function of human carbonic anhydrase I (hCA-I). Using kinetic studies, highresolution mass spectrometry, and NMR analysis, we observed changes in protein structure and function depending on the protein and cross-linker concentrations. Systematic examination of various conditions enabled us to outline general reaction conditions to preserve the structural and functional integrity of protein during cross-linking. EXPERIMENTAL SECTION Chemicals and Materials. Unless stated otherwise, all chemicals (including proteins) were purchased from SigmaAldrich (St. Louis, MO). Acetonitrile and water were from Merck (Darmstadt, Germany). Protein production. The full-length coding region of hCA-I (UniProt: P00915) was synthesized by Shanghai Generay

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Biotech and subcloned into pET-21a(+). For the protein production, the expression plasmid was introduced into E. coli BL21(DE3) Gold strain (Stratagene) by transformation. Large-scale protein production of unlabeled or 15N labeled hCA-I was carried out in LB or M9 medium, respectively, containing 15NH4Cl (Cambridge Isotope Laboratories) as a sole nitrogen source. When the cell density reached OD600 of 0.8, overexpression of hCA-I was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside and 0.5 mM ZnSO4. After induction, cells were grown for 12 h at 25°C and then were harvested by centrifugation at 6,000× g for 10 min. The pellet was resuspended in a lysis buffer containing 50 mM Na2HPO4 (pH 7.5), 50 mM NaCl, 1 mM NaN3, 1 mM PMSF, and lysozyme (250 µg/ml) and kept for 15 min at room temperature. The cells were lysed by sonication on ice for 5 min and then supplemented with 100 µg DNAse I and 400 µl of 1 M MgCl2. The cell extract was centrifuged at 45,000 × g for 15 min, and the supernatant was used for the purification of hCA-I by the IMAC chromatography using Cu2+ ions as described previously33. For IMAC purification, the Ni Sepharose 6 FF (GE Healthcare) was packed onto in a 10/100 column, the Ni2+ ions were removed, and the medium was recharged with Cu2+ ions. The supernatant was loaded onto the column and washed with the buffer containing 50 mM Na2HPO4 (pH 7.5), 50 mM NaCl, 1 mM NaN3, and 1 mM imidazole. The bound enzyme was eluted by linear gradient of imidazole from 1 to 200 mM. The fractions containing hCA-I were pooled and purified by gel filtration on Superdex 200 10/300 GL column (GE Healthcare). Protein concentration was measured by Bradford assay (Bio-Rad) with commercial hCA-I as a standard. Chemical cross-linking. To investigate the concentration dependence of cross-linker on protein structure, hCA-I was cross-linked in a buffer containing 15 mM HEPES (pH 7.5) and 150 mM NaCl at different concentrations of bis(sulfosuccinimidyl)suberate(BS3) or bis(sulfosuccinimidyl)glutarate(BS2G) (ProteoChem) with 5 to 500 molar excess. The enzyme concentration was set to 0.2 mg/ml (6.9 μM). To examine the concentration dependence of protein on protein structure, hCA-I was cross-linked at 10 molar excess of BS3 or BS2G at different concentrations of the enzyme such as 0.2 mg/ml, 1.0 mg/ml, and 2.0 mg/ml. The cross-linking reaction was allowed to proceed for two hours at room temperature. The reaction in the absence of cross-linking reagent was carried out as a control. After the cross-linking reaction, the enzyme activity was measured in each sample, and the reaction mixtures were analyzed by SDS-PAGE and high-resolution mass spectrometry. For chemical cross-linking of alcohol dehydrogenase 1 ADH, we used the same conditions as for hCA-I experiment (described above) with one exception. Since the optimum ADH activity is at pH 8.5, we increased the pH of buffer (15 mM HEPES and 150 mM NaCl) from 7.5 to 8.5. Enzyme activity. The esterase activity of cross-linked and unmodified hCA-I was measured spectrophotometrically with 4-nitrophenyl acetate (Sigma-Aldrich) as a substrate. In the measurement, 20 µl of hCA-I was mixed with 80 µl of freshly prepared 12 mM 4-nitrophenyl acetate in 15 mM HEPES (pH 7.5), 150 mM NaCl, and 10% acetonitrile. The increase in absorption at 348 nm was measured in triplicate in a96-well microtiter plate at 25°C. The dehydrogenase activity of cross-linked and unmodified ADH was measured spectrophotometrically with β nicotinamide adenine dinucleotide (NAD) and ethanol (Thermo Fisher Scientific) as substrates. All samples including control were diluted to the concentration of 0.2

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µg/ml by 100 mM phosphate buffer (pH 8.5). During measurement, 10 µl of diluted ADH was mixed with 10 µl of freshly prepared 10 mM NAD, 20 µl of 1M ethanol, and 160 µl of 100 mM phosphate buffer (pH 8.5). The increase in absorption at 340 nm was measured in triplicate in a 96well microtiter plate at 25°C. Accurate measurement of intact protein mass. Crosslinked and unmodified hCA-I samples were desalted on MicroTrap column and analyzed by direct fusion on ApexUltra Qe Fourier transformed mass spectrometer equipped with a 9.4 T superconducting magnet. Mass spectra were obtained by accumulating ions in the ESI source hexapole and running the Q mass filter that allowed only ions of broad m/z range (300-2000) to pass through the FT-MS analyzer cell. Data acquisition and data processing were performed with ApexControl 3.0.0 and DataAnalysis 4.0. Mass spectrometric identification of cross-links. For the cross-linking, BS3 or BS2G were used as 1:1 mixtures of nondeuterated and four times deuterated derivates ito facilitate identification of cross-links. Identification of crosslinks was performed as described previously13, 34. Briefly, SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the reaction mixture was accomplished, and bands (10 and 100 molar excesses) of the cross-linked enzymes were excised. In gel proteolysis by trypsin was carried out overnight. For peptides quantitation, only nondeuterated BS3 or BS2G was used in 10 and 100 molar excesses. After two hours of incubation, in solution trypsin digestion (final enzyme:protein ratio 1:20 w/w) was performed directly in the reaction mixture. The volume of peptide mixtures corresponding to 100 ng of initial protein was injected and subsequently desalted on reversed-phase trap column (Agilent, Zorbax 300SB-C18 0.3 ×5 mm, 5 µm). The eluted peptides were separated on the reversed-phase analytical column (Agilent, Zorbax 300SB-C18 0.3 ×150 mm, 3.5 µm) at 40°C using an Agilent 1200 HPLC system. The system was directly coupled to the solariX XR FT-ICR mass spectrometer (Bruker Daltonics) equipped with a 15T superconducting magnet. Mass spectral data were collected in positive broadband mode over the m/z range 250–2500, with 1M data points transient and 0.2 s ion accumulation with two averaged scans per spectrum. Data acquisition and data processing were performed using ftmsControl 2.1.0 and DataAnalysis 4.4. The cross-linked peptides were identified using Links software1. Data acquisition of quantitative samples was realized in technical triplicate. Absolute peptide intensity values were deducted as the maximal intensity of the monoisotopic peak abundance in the chromatogram. NMR spectroscopy. 1H-15N TROSY spectra were recorded at 30 °C on BrukerAvance III 600 MHz spectrometer equipped with the TCI cryoprobe. NMR experiments were carried out using uniformly labeled 15N protein sample of 10 mg/ml concentration in 40 mM phosphate buffer (pH 6.2) and 10% D2O. NMR data were processed using the NMRPipe35 and analyzed using the Sparky36 software. For analysis of changes in 1H-15N TROSY spectra, combined chemical shifts (Δδ) expressed as Δδ=(25 Δδ(1H)2 + Δδ(15N)2)1/2, were calculated for the 1H and 15N backbone resonances, where Δδ(1H) and Δδ(15N) are the differences between shifts of unmodified and cross-linked hCA-I, respectively. RESULTS AND DISCUSSIONS Chemical cross-linking affects protein function. Optimal protein and cross-linker concentrations in coupling reactions are protein specific and depend on the protein size and the number of accessible reactive groups. For BS3 and BS2G,


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which are the most commonly used cross-linkers, the optimal conditions were usually kept at micromolar protein concentration and 20 to500-fold molar excess of crosslinker37. To investigate the effect of cross-linking on protein structure and function, we cross-linked human carbonic anhydrase I (CA-I) using different concentrations of BS3 or BS2G. hCA-I was chosen because its three-dimensional structure, NMR backbone resonance assignment, and enzyme activity are well characterized38,39. First of all, hCA-I was cross-linked at a constant protein concentration of 0.2 mg/ml (6.9 μM) by different concentrations of cross-linker ranging from 5 to 500 molar excess and all samples were analyzed by SDS-PAGE and mass spectrometry. Monolinks and cross-links were determined for both cross-linkers and molar excesses (Table S1 and S2). Surprisingly, no gain of identified cross-links was observed when samples with 10 and 100 molar excesses of cross-linkers were compared. Aggregation or oligomerization of hCA-I caused by excessive cross-linking was not observed for any of the used cross-linkers (Figure 1a, b). However, a band with slightly lower molecular weight than hCA-I was detected at higher concentrations of cross-linkers. The observation of the band with apparently lower molecular weight indicates a change in electrophoretic mobility of hCA-I, which is likely due to more compact protein arrangement caused by excessive cross-linking. In the next step, we focused on the kinetic studies to investigate the effect of cross-linker on the enzymatic activity. hCA-I was cross-linked as described earlier, and the enzyme activity was measured for each sample and compared to the enzyme activity of the unmodified enzyme.

With increasing concentrations of BS3, the enzyme activity significantly decreased (Figure 1c) and dropped down to only 50% of the initial activity of the unmodified enzyme at 500 molar excess of cross-linker. On the other hand, increasing BS2G concentrations surprisingly led to an increase in hCA-I relative activity (up to 108%) (Figure 1d), which subsequently decreased to 77% at 500 molar excess of cross-link. This observation simply implies that protein function is affected by cross-linking in spite of the fact that SDS-PAGE, which is usually used for optimization of reaction conditions, did not reveal any significant change in the electrophoretic mobility. The effect on protein function is dependent on cross-linker concentration and is extensive for heavily cross-linked protein. We further examined the effect of cross-linking on the structure and function of yeast ADH, which is a 141 kD noncovalent tetramer containing four equal subunits. ADH was cross-linked by BS3 or BS2G under the same conditions as in the case of hCA-I. The SDS-PAGE analysis revealed that the ADH tetramer is cross-linked only at higher cross-linker concentrations, whereas use of lower cross-linker concentrations resulted in lower oligomeric states (Figure S1a, b). The measurement of the enzyme activity of ADH upon crosslinking showed a similar effect to that observed in the case of hCA-I. Like hCA-I activity, the ADH activity also decreased significantly with increase in cross-linker concentrations, and the effect was more extensive for the longer cross-linker BS3 (Figure S1c, d). Moreover, mass spectrometric analysis of cross-linking products revealed the same yield of monolinks and crosslinks at both cross-linker concentrations as well (Table S3 and S4).

Figure 1.The effect of cross-linker concentration on protein structure and function. Enzymatic activity assay of hCA-I after cross-linking by different concentrations of BS3 (a) or BS2G (b). SDS-PAGE of reaction products after hCA-I cross-linking by increasing concentration of BS3 (c) and BS2G (d). In all cases, hCA-I concentration was kept at 0.2 mg/mL (6.9 μM). BS3 and BS2G affect protein structure in a different way. Previous kinetic measurements showed that the enzyme activities of hCA-I and ADH upon cross-linking depends not only on the cross-linker concentration but also on the size of the cross-linking reagent. As both BS3 and BS2G represent the same type of cross-linking reagent with almost the same reactivity and they differ from each other

only in the length of the spacer arm (11.4 Å for BS3 and 7.7 Å for BS2G), their remarkably different effects on the enzyme activity might be explained by different products of cross-linking. In principle, the cross-linking by an amine-amine bifunctional cross-linker can lead to the formation of two possible products: cross-links (Type 1 and 2) and monolinks



(Type 0). While in the cross-links two spatially proximate residues of protein are converted into covalent bond by cross-linker, in monolinks only one reactive group of crosslinker interacts with the protein, and the other is hydrolyzed because it does not come into contact with other crosslinkable residues34,40. Therefore, cross-linking by shorter BS2G might produce more monolinks in comparison with the longer BS3. To verify the previous hypothesis, the accurate mass of hCA-I upon cross-linking by 5, 10, 50 and 100 molar excess of cross-linker was measured by high-resolution mass spectrometry. The mass spectra as shown in Figure 2 and Figure S2 revealed that the amount of unmodified hCA-I decreases with increasing concentrations of cross-linker, whereas the amount of cross-linked products increases. At 50 molar excess of cross-linker, the peak corresponding to the unmodified protein is not detected, and masses are shifted to higher values corresponding to the products with two and more cross-links and monolinks. At 100 molar

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excess of cross-linker, the reaction mixture contained so many products that we were unable to identify all of them. As we already proposed, there is a difference in the abundance ratio between cross-links and mono-links for BS3 and BS2G. This can be observed in mass spectra when lower cross-linker concentrations were used. Detailed inspection of the spectra in Figure 2 and Figure S2 indicated that at 5 and 10 molar excess of cross-linker the ratio between the protein with one cross-link and protein with one monolink is approximately 1:1 for BS3, whereas it is approximately 1:2 for BS2G. This observation is consistent with the previous hypothesis that in many cases the shorter cross-linker BS2G cannot reach another lysine in the protein and its reactive group hydrolyzes to form monolink. In contrast, the longer cross-linker BS3 forms more cross-links because it can come into contact with more lysine residues. However, the question remains as to how cross-links

Figure 2. Overview of the mass spectra of unmodified hCA-I and hCA-I cross-linked by 5, 10, 50 and 100 molar excess of BS2G. The products of cross-linking containing cross-links and mono-links are shown above and below spectra.


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and monolinks affect protein structure and function. We could consider mainly two effects: changes in protein flexibility and disruption of the electrostatic interactions caused by the replacement of a positive charge of lysine with a cross-link or monolink carrying negative charge on it. Also, mass spectra of hCA-I at low cross-linker concentration, which preserves the structural integrity of protein, reveal a low yield of the reaction. Thus, the choice of cross-linker concentration is a compromise between the reaction yield and an acceptable structural distortion. Previous CXMS studies have used cross-linking under single-hit conditions which would allow each molecule to be cross-linked only once10,41. However, as we described here, in practice it is often difficult to achieve the single-hit conditions because cross-linking even at low cross-linker concentration leads to a heterogeneous mixture containing several products with various numbers of cross-links and monolinks. Furthermore, when a protein complex is crosslinked, much higher cross-linker concentration exceeding single-hit conditions needs to be used to cross-link all subunits. Cross-linking does not affect protein structure significantly at low cross-linker concentration. To probe structural changes induced by cross-linking, we performed NMR measurements of cross-linked hCA-I. In this approach, the chemical shift changes of the backbone amide were monitored by measuring the 1H-15N TROSY. Recombinant 15N-labeled hCA-I was prepared in bacterial expression system, and 1H-15N TROSY spectrum was measured at the protein concentration of 10 mg/ml. Then the protein was diluted to the concentration of 0.2 mg/ml and was cross-linked by 10 or 100 molar excess of BS3 or BS2G. After cross-linking, the protein concentration was adjusted to10 mg/ml, then the 1H-15N TROSY spectrum was

measured and compared to the spectrum of unmodified hCA-I. Superposition of the 1H-15N TROSY spectra of the unmodified and cross-linked hCA-I revealed new crosspeaks reflecting the heterogenous mixture of differently crosslinked or monolinked hCA-I (48 new cross-peaks for the 10 molar excess of BS3, 84 new cross-peaks for the 100 molar excess of BS3, 59 new cross-peaks for the 10 molar excess of BS2G, and 125 new cross-peaks for the 100 molar excess of BS2G), as well as changes in relative intensities and the peak positions of several primary cross-peaks (Figure S3 and S4). To quantify structural perturbations induced by cross-linking, we calculated combined chemical shifts (Δδ) which is a very sensitive parameter for monitoring structural and functional changes. As long as a protein has been changed by a certain chemical event, the NMR peaks may change their position, shape, intensity, or disappear because of the changed local environment. If the local environment does not change, then no chemical shift perturbation will be seen. The Δδ was calculated for each backbone cross-peak (Figure 3) with the exception of those that could not be assigned due to overlap or a significant decrease in the cross-peak intensity. In the case of intensity change, an arbitrary value of Δδ was set to 2.0 ppm. The arbitrary value of 2.0 ppm has been chosen to clearly illustrate the residues, which are affected by extensive changes in the local chemical environment. Upon crosslinking by BS3, the largest perturbations with the Δδ>0.5 ppm were observed for 12 residues at 10 molar excess of cross-linker and for 61 residues at 100 molar excess of cross-linker. Regarding BS2G, the limit Δδ>0.5 ppm was exceeded by 15 residues at 10 molar excess of BS2G and 51 residues at 100 molar excess of cross-linker. Intriguingly, BS3 affects a different set of residues than BS2G (Figure 3).

Figure 3.The plot of average chemical shift perturbations (Δδ) of hCA-I backbone amides against residue number upon crosslinking by 10 and 100 molar excesses of BS2G or BS3. The bars are colored in the gradient ramping up from white (low Δδ values) to red (high Δδ values) to show the most perturbed residues. The negative bars indicate proline residues or residues that could not be unambiguously assigned due to overlap. For the residues with a significant change of the cross-peak intensity, the arbitrary value was set at 2.0 ppm. Monolinks (●) and cross-links (× or +) were identified by mass spectrometry.



Taken together, NMR analysis demonstrated that more structural perturbations are induced at higher cross-linker concentration, and the effects of BS3 and BS2G on the structure of hCA-I are different. The differential effects of BS3 and BS2G on the protein structure is likely based on the fact that the shorter BS2G might produce more monolinks than the longer BS3, as we have shown in this study. However, we were unable to decipher the particular mechanism of BS3 or BS2G action using the calculated chemical shifts perturbations. A vast majority of backbone cross-peaks were rather slightly affected and only local perturbations are apparent, suggesting that cross-linking induces mainly local structural disturbances. Even at high cross-linker concentration, we have not observed extensive structural perturbations in regions corresponding to the secondary structural elements indicating that the overall fold is broadly preserved. However, this fact might be specific for carbonic anhydrase as it does not show high dynamic motions and is largely well structured. This effect might be different for dynamic proteins with large unstructured and flexible regions or intrinsically disordered state. We further investigated the correlation between the residues which are structurally most affected by crosslinking and the enzyme activity of hCA-I. The decrease in enzyme activity appeared to be as a result of structural changes in the architecture of the binding cavity. Indeed, upon cross-linking at 100 molar excess of BS3, severe perturbations in chemical shift have been detected for residues such as H200, F91, or A121, which are building blocks of the binding cavity of hCA-I. Factors affecting the structural changes induced by cross-linking. hCA-I has 18 lysine residues and each lysine

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residue likely affects the protein structure in a different way. We further examined the correlation between individual lysine residue and structural perturbation of hCA-I by using NMR study. First, we cross-linked hCA-I with 10 and 100 molar excesses of BS3 or BS2G and identified both crosslinks and monolinks by high-resolution mass spectrometry. All unique cross-links and monolinks are listed in TableS1 and TableS2, respectively, and are depicted in the plot of Δδ values (Figure 3) calculated from the previous NMR experiment. Intriguingly, a few lysine residues, which are involved in cross-links or monolinks, are severely perturbed while the others are perturbed slightly or not at all (Figure 4 and Figure S5). Moreover, several lysine residues induce significant perturbations of neighboring residues upon crosslinking. Apart from cross-links between two lysine residues, unexpected side reactions have been revealed in recent studies with NHS esters showing the formation of crosslinks between lysine and hydroxyl containing amino acids4245 . Thus, we searched for Ser/Thr/Tyr–Lys crosslinks in the LC-MS spectra and identified S77-K80 cross-link and S228 monolink. Interestingly, both S77 and S228 are in close proximity with an arginine, which has been shown to increase reactivity to NHS esters46. To find the correlation between individual lysine residues and structural perturbation, for each lysine residue, we calculated an average B-factor of Cα atom, an average number of Nζ salt bridges, an average number of Nζ hydrogen bonds, and an average Nζ accessible surface area (Fig. S6) for 36 hCA-I structures available in the PDB database (PDB IDs: 1azm, 1bzm, 1crm, 1czm, 1hcb, 1hug, 1huh, 1j9w, 1jv0, 2cab, 2foy, 2fw4, 2it4, 2nmx, 2nn1, 2nn7, 3lxe, 3w6h, 3w6i, 4wr7, 4wup, 4wuq). Comparison of the

Figure 4. Structural changes of hCA-I mapped onto the crystal structure upon cross-linking with 10 molar excess (a) and 100 molar excess (b) of BS2G. Residues are colored as in Fig. 3. The zinc atom in the active site is colored blue.


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average number of salt bridges and Δδ calculated from NMR study revealed that lysine residues with an absence or a small number of salt bridges show similar Δδ – low values for 10 molar excess of cross-linker and very high values for 100 molar excess of cross-linker. Furthermore, residues in the close vicinity of those lysine residues show a significant increase in Δδ suggesting a structural perturbation in the local environment. In contrast, the lysine residues with a relatively high number of salt bridges (K57, K127, K213) were observed with low values of Δδ for both 10 and 100 molar excess of BS3 or BS2G. However, the numbers of salt bridges did not seem to always correlate with Δδ. For example, K156 with the absence or K34 with the relatively high number of salt bridges failed to share the common behavior as described earlier. Next, we assessed the relative reactivity of first lysine residues with the structural perturbations. To determine relative reactivity of lysine residues, we compared the intensity of individual modified or unmodified peptides at 10 and 100 molar excesses of cross-linker (Fig. S7). The increase of cross-linker from 10 to 100 molar excess led to a drop in the intensity of the unmodified peptides and, on the other hand, to a rise in the intensity of the modified peptides. Even though the cross-linker concentration increased 10 times, we did not observe 10 times change in the intensity of modified or unmodified peptides possibly due to a higher complexity of products. Detailed analysis of the intensity of peptides revealed that K45, K10, and K80 are most reactive among the first 7 lysine residues. In contrast, K34 appeared to be the less reactive lysine residue. Intriguingly, using NMR study, we observed that the reactivity for first 7 lysine residues correlated with the solvent accessible surface area (Fig. S6D), the average numbers of salt bridges (Fig.S6B), and finally with Δδ. Moreover, such analysis pointed out an interesting behavior. NMR spectra demonstrated that the most reactive lysine residues induce the high increase of Δδ while the less reactive residues induce the increase of Δδ of the surrounding regions. However, it is noteworthy that the effect of cross-linking on the protein structure is probably more complex issue influenced by many other factors– besides the salt or hydrogen bonding interactions, or solvent accessible area it might be due to protein flexibility which we did not observe in this study as hCA-I does not show regions with high dynamic motions. The local electrostatic environment can also play a role in affecting protein structure by influencing the effective pKa, and hence the reactivity of lysine side-chains. The identified cross-links are in good agreement with published high-resolution structures of hCA-I. To verify the accuracy of determined Lys-Lys cross-links, the solvent accessible surface (SAS) distances between LysNζ atoms up to 50 Å were calculated using program Xwalk47 for 36 structures of hCA-I structures available in the PDB database as described above. The majority of experimentally determined SAS distances between Nζ of cross-linked lysines is ≤23.1 Å for BS3 and ≤19.4 Å for BS2G, respectively (Figure S8). The boundary SAS distance of 23.1 Å for the BS3 cross-linker corresponds to the length of the BS3 spacer (11.4 Å) plus 11.7 Å reflecting the lysine side-chain flexibility. In a similar way, the boundary SAS distance was set at 19.4 Å (7.7 Å plus 11.7 Å) for the BS2G cross-linker. For BS3 cross-linker only two cross-links (K39-K45 and K168-K170) exceeded maximum distance by 3.2 Å and 9.8 Å, respectively. In the case of BS2G crosslinker, the maximum distance was above the limit for three cross-links (K34-K45, K39-K45 and K168-K170) by

22.9 Å, 6.9 Å, and 13.5 Å, respectively. Residues K39 and K45 are located in the region with slightly higher average Bfactors of Cα atoms calculated from X-ray structures (Figure S9), which can reflect the higher flexibility of this region and thus the possibility of the cross-link establishment. Similarly, the higher Cα B-factors are in the loop formed by residues L230-P240 which covers the residues K168 and K170. Higher flexibility of the loop can increase the accessibility of the cross-linker and induce cross-link formation. In addition, the higher flexibility of both regions was also predicted from backbone chemicals shifts, deposited in the BMRB database, by program TALOS+48. hCA-I tends to form a dimer during cross-linking at high protein concentration. The next important factor which might have an effect on protein structure during crosslinking is the protein concentration. In principle, crosslinking at higher protein concentration might form artificial oligomers. Thus, we decided to investigate the relationship between the protein concentration and the enzymatic activity as well as oligomer formation during chemical crosslinking. In this experiment, the hCA-I was cross-linked at different protein concentrations such as 0.2, 1.0, and 2.0 mg/ml with constant 10 molar excess of cross-linker. The 10 molar excess of cross-linker was chosen due to the satisfactory yield and little effect on protein structure as already shown. Comparison of enzyme activities of crosslinked and unmodified enzyme (Figure 5a,b) showed that upon cross-linking by BS3, the enzyme activity decreased to 80% and 71% at the protein concentration of 0.2 mg/ml and 1.0 mg/ml, respectively, and then remained at a constant level of 2.0 mg/ml.

Figure 5. The protein concentration dependence on protein structure in chemical cross-linking. The hCA-I was crosslinked at 10 molar excess of BS3 or BS2G and different concentrations of enzyme. Relative enzyme activity of hCAI was measured spectrophotometrically after cross-linking by BS3 (a) or BS2G (b) and normalized per concentration of unmodified enzyme (wt). The mixtures of hCA-I after crosslinking by BS3 (c) or BS2G (d) were analyzed by SDSPAGE.



On the other hand, after cross-linking by BS2G, the enzyme activity remained unchanged and reached a plateau for all tested protein concentrations. The reaction mixtures were further analyzed by SDSPAGE. For both cross-linkers, a band with slightly lower molecular weight and a band corresponding to the dimer of hCA-I were observed at protein concentrations of 1.0 and 2.0 mg/ml (Figure 5c,d), respectively. The artificial dimerization of hCA-I is likely a result of higher protein concentration in which the chances of protein-protein collision are greater and therefore more collisions can be converted into covalent dimer by cross-linkers. This observation suggests that protein concentration is another important factor affecting protein structure and oligomerization state during cross-linking; and to preserve the structural and functional integrity of protein, the crosslinking should be performed at a low protein concentration. On the other hand, the low protein concentration is far from physiological conditions as the cellular interior is filled with macromolecules at very high concentrations where the proteins are entropically driven towards a stabilized conformation using the molecular crowding effect49. CONCLUSION CXMS offers a simple and fast way to study the protein structure and the protein-protein interactions; however, this method requires careful consideration. Here we describe how different factors such as the type of cross-linker, protein concentration, or cross-linker concentration can affect protein structure and function. Using kinetic measurements and NMR analysis we demonstrated that functional and structural integrity of protein is mostly affected at higher cross-linker concentration. Use of high cross-linker concentration affects the protein structure rather locally, and the protein fold remains unchanged. However, even such small structural changes result in a significant drop in enzyme activity. On the other hand, there is no significant yield of identified cross-links using higher concentrations of cross-linker resulting in better spatial resolution for both enzymes. Thus, it is sufficient to use the molar excess of cross-linker equal to the number of reactive residues. These findings nicely correlate with previously published data showing the yield of the cross-linking reaction is more as determined by the reactivity and accessibility of lysine residues not by the concentration of cross-linker. Protein concentration is a further significant parameter that requires careful consideration in chemical cross-linking. As we have shown using the kinetic measurements, higher protein concentration resulted in lower enzyme activity. Moreover, higher protein concentration tends to form dimers. Hence, in order to preserve the native structure of protein during cross-linking, the cross-linker, as well as the protein concentration, should be used at a low level. On the other hand, such experimental conditions tend to produce very low yields and the low protein concentration is usually a condition far from the crowded environment inside the cell. Thus, the choice of cross-linker concentration is a compromise between the reaction yield and an acceptable structural distortion. Analysis of products after cross-linking using SDS-PAGE has been shown to be very useful as the bands corresponding to oligomers or bands with different electrophoretic mobility than the unmodified protein reflected heavy structural changes. However, unlike NMR spectroscopy, SDS PAGE analysis of cross-linking products does not reveal the local structural perturbations or changes in protein function.

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The next important aspect of cross-linking is the crosslinker selection. The shorter cross-linkers lead to the formation of monolinks while longer cross-linkers produced more cross-links. Moreover, the effects of monolinks and cross-links on protein structure are different as described via NMR study and are probably caused by disruption of electrostatic interactions. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Tables of identified cross-links and monolinks upon crosslinking of hCA-I and ADH with 10 and 100 molar excesses of BS3 or BS2G.The effect of cross-linker concentration on function of ADH. Superposition of the 1H-15N TROSY spectra of unmodified hCA-I, hCA-I cross-linked by 10 molar excess of BS3/BS2G) and hCA-I cross-linked by 100 molar excess of BS3 BS3/BS2G. Structural changes of hCA-I mapped onto the crystal structure upon cross-linking with 10 and 100 molar excess of BS3. The average B-factor of Cα atom, the average number of Nζ salt bridges, the average number of Nζ hydrogen bonds and the average Nζ accessible surface area for each lysine residue of hCA-I. The intensity of unmodified and modified peptides upon crosslinking with BS3 or BS2G determined for first 7 lysine residues. The monolinks and cross-links identified by mass spectrometry after cross-linking of hCA-I. Residue fluctuation. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Fax: +420 241 062 156 (PN) Tel.: +420 325 873 610 (PN). Author Contributions The manuscript was written through contributions of all authors. All authors approved the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the Czech Science Foundation (grant numbers 16-24309S), the Ministry of Education of the Czech Republic (projects LH15010, LD15089; programs “NPU I” project LO1509 and “NPU II” project LQ1604; LM2015043 CIISB for CMS BIOCEV; LTC17065), COST Action (BM1403), BIOCEV (grant number ERDF CZ.1.05/1.1.00/02.0109), and in part by the Czech Academy of Sciences (grant number RVO61388971), and by the Charles University in Prague (grant numbers UNCE 204025/2012 and GACU 932316). ABBREVIATIONS CXMS, Chemical cross-linking coupled with mass spectrometry; BS3, bis(sulfosuccinimidyl)suberate; BS2G, bis(sulfosuccinimidyl)glutarate; hCA-I, human carbonic anhydrase I; ESI, electrospray ionization; FT-ICR, Fouriertransformed ion cyclotron resonance; MS, mass spectrometry; ADH, alcohol dehydrogenase 1 from Saccharomyces cerevisiae; NAD, β nicotinamide adenine dinucleotide


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Figure 1. The effect of cross-linker concentration on protein structure and function. Enzymatic activity assay of hCA-I after cross-linking by different concentrations of BS3 (a) or BS2G (b). SDS-PAGE of reaction products after hCA-I cross-linking by increasing concentration of BS3 (c) and BS2G (d). In all cases, hCA-I concentration was kept at 0.2 mg/mL (6.9 µM). 117x83mm (300 x 300 DPI)



Figure 2. Overview of the mass spectra of unmodified hCA-I and hCA-I cross-linked by 5, 10, 50 and 100 molar excess of BS2G. The products of cross-linking containing cross-links and mono-links are shown above and below spectra. 213x223mm (300 x 300 DPI)


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Figure 3. The plot of average chemical shift perturbations (∆δ) of hCA-I backbone amides against residue number upon cross-linking by 10 and 100 molar excesses of BS2G or BS3. The bars are colored in the gradient ramping up from white (low ∆δ values) to red (high ∆δ values) to show the most perturbed residues. The negative bars indicate proline residues or residues that could not be unambiguously assigned due to overlap. For the residues with a significant change of the cross-peak intensity, the arbitrary value was set at 2.0 ppm. Monolinks (●) and cross-links (× or +) were identified by mass spectrometry. 177x112mm (300 x 300 DPI)



Figure 4. Structural changes of hCA-I mapped onto the crystal structure upon cross-linking with 10 molar excess (a) and 100 molar excess (b) of BS2G. Residues are colored as in Fig. 3. The zinc atom in the active site is colored blue. 148x108mm (300 x 300 DPI)


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Figure 5. The protein concentration dependence on protein structure in chemical cross-linking. The hCA-I was cross-linked at 10 molar excess of BS3 or BS2G and different concentrations of enzyme. Relative enzyme activity of hCA-I was measured spectrophotometrically after cross-linking by BS3 (a) or BS2G (b) and normalized per concentration of unmodified enzyme (wt). The mixtures of hCA-I after cross-linking by BS3 (c) or BS2G (d) were analyzed by SDS-PAGE. 90x109mm (300 x 300 DPI)



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Impact of Chemical Cross-Linking on Protein Structure and Function

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