Impact of Chemical Cross-Linking on Protein Structure and Function

Article pubs.acs.org/ac

Cite This: Anal. Chem. 2018, 90, 1104−1113

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, 14220 Prague, Czech Republic Department of Biochemistry, Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic

‡

S Supporting Information *

ABSTRACT: Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on 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.

C

of protein conformation and protein interactions.26−30 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 determination.31,32 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 cross-linker. 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 a 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, high-resolution mass spectrometry, and NMR analysis, we observed changes in protein structure and function depending on the protein and

hemical 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 cryo-electron 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 other.2 The cross-linked protein is then digested, and the resulting peptide mixture is analyzed by mass spectrometry in order to identify cross-linked peptides.3 Cross-link derived constraints are ultimately used for model building.4−6 The popularity of CXMS has increased steadily, and a significant progress has been made in applications, in new protocols, and in improvement of bioinformatic tools in recent years.7−14 The CXMS approach was successfully applied in structural analyses of various challenging macromolecular complexes such as RNA polymerase,15,16 prokaryotic ribosome,17 TriC/CCT chaperonin,18,19 proteasome,20−24 and membrane proteins.25 Furthermore, recent advances in quantitative CXMS pave the way toward analyzing dynamics © 2017 American Chemical Society

Received: July 21, 2017 Accepted: December 12, 2017 Published: December 12, 2017 1104

DOI: 10.1021/acs.analchem.7b02863 Anal. Chem. 2018, 90, 1104−1113

Article

Analytical Chemistry

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 a 96-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 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 1 M 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 96-well microtiter plate at 25 °C. Accurate Measurement of Intact Protein Mass. Crosslinked and unmodified hCA-I samples were desalted on a MicroTrap column and analyzed by direct fusion on an ApexUltra Qe Fourier transformed mass spectrometer equipped with a 9.4 T superconducting magnet. Mass spectra were obtained by accumulating ions in the electrospray ionization (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-mass spectrometry (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 was used as a 1:1 mixture of nondeuterated and four times deuterated derivates to facilitate identification of cross-links. Identification of cross-links was performed as described previously.13,34 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 2 h 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 a 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 Fourier-transformed ion cyclotron resonance (FTICR) mass spectrometer (Bruker Daltonics) equipped with a 15T superconducting magnet. Mass spectral data were collected in positive broadband mode over the m/z range of 250−2500, with 1 M 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 software.1 Data acquisition of quantitative samples was realized in technical triplicate. Absolute peptide intensity

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 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 an 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 6000g 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 of DNase I and 400 μL of 1 M MgCl2. The cell extract was centrifuged at 45 000g for 15 min, and the supernatant was used for the purification of hCA-I by the IMAC chromatography using Cu2+ ions as described previously.33 For IMAC purification, the Ni Sepharose 6 FF (GE Healthcare) was packed onto 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 the 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(sulfo-succinimidyl)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, 1.0, and 2.0 mg/mL. The cross-linking reaction was allowed to proceed for 2 h 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 from Saccharomyces cerevisiae (ADH), we used the same conditions as for hCA-I experiment (described above) with one exception. 1105


Article


Figure 1. 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).

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 kDa 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

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 a 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 hCAI, respectively.

■

RESULTS AND DISCUSSION

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, which are the most commonly used cross-linkers, the optimal conditions were usually kept at micromolar protein concentration and 20- to 500-fold molar excess of crosslinker.37 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 characterized.38,39 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 a 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 (Tables S1 and S2). 1106


Article


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 monolinks are shown above and below spectra.

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 (Types 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 cross-linker interacts with the protein, and the other is hydrolyzed because it does not come into contact with other cross-linkable residues.34,40 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 hCAI upon cross-linking by 5, 10, 50, and 100 molar excess of cross-linker was measured by high-resolution mass spectrom-

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 an 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 cross-links at both cross-linker concentrations as well (Tables S3 and S4). 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 1107


Article


Figure 3. Plot of average chemical shift perturbations (Δδ) 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 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.

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 crosslinker 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 once.10,41 However, as we described here, in practice, it is often difficult to achieve the single-hit conditions because crosslinking 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 cross-linked, much higher crosslinker 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 the bacterial expression system, and the 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.

etry. The mass spectra as shown in Figures 2 and 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 crosslinker, 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 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 monolinks for BS3 and BS2G. This can be observed in mass spectra when lower cross-linker concentrations were used. Detailed inspection of the spectra in Figures 2 and 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 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 1108


Article


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 Figure 3. The zinc atom in the active site is colored blue.

After cross-linking, the protein concentration was adjusted to 10 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 cross-peaks reflecting the heterogeneous mixture of differently cross-linked 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 (Figures 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, or 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 cross-linking 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 of 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). 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 an intrinsically disordered state. We further investigated the correlation between the residues which are structurally most affected by cross-linking and the enzyme activity of hCA-I. The decrease in enzyme activity appeared to be as a result of structural changes in the 1109


Article


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, we observed that the reactivity for the first 7 lysine residues correlated with the solvent accessible surface area (Figure S6D), the average numbers of salt bridges (Figure 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 a more complex issue influenced by many other factors; besides the salt or hydrogen bonding interactions or the 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. 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 crosslinks (K39−K45 and K168−K170) exceeded the maximum distance by 3.2 and 9.8 Å, respectively. In the case of BS2G cross-linker, the maximum distance was above the limit for three cross-links (K34−K45, K39−K45, and K168−K170) by 22.9, 6.9, and 13.5 Å, respectively. Residues K39 and K45 are located in the region with slightly higher average B-factors 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, cross-linking 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 cross-linking. 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 a constant 10 molar excess of cross-linker. The 10 molar excess of crosslinker was chosen due to the satisfactory yield and little effect on protein structure as already shown. Comparison of enzyme activities of cross-linked and unmodified enzyme (Figure 5a,b)

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 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 an NMR study. First, we cross-linked hCA-I with 10 and 100 molar excesses of BS3 or BS2G and identified both cross-links and monolinks by high-resolution mass spectrometry. All unique cross-links and monolinks are listed in Tables S1 and S2, 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 (Figures 4 and S5). Moreover, several lysine residues induce significant perturbations of neighboring residues upon cross-linking. Apart from cross-links between two lysine residues, unexpected side reactions have been revealed in recent studies with NHS esters showing the formation of cross-links between lysine and hydroxyl containing amino acids.42−45 Thus, we searched for Ser/Thr/ Tyr−Lys cross-links 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 esters.46 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 (Figure 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, 2 nmx, 2nn1, 2nn7, 3lxe, 3w6h, 3w6i, 4wr7, 4wup, 4wuq). Comparison of the average number of salt bridges and Δδ calculated from the 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 crosslinker. 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 (Figure 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 a 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 1110


Article


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 determined by the reactivity and accessibility of lysine residues and not by the concentration of cross-linker.50,51 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 crosslinking, 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. The next important aspect of cross-linking is the cross-linker selection. The shorter cross-linkers lead to the formation of monolinks while longer cross-linkers produced more crosslinks. Moreover, the effects of monolinks and cross-links on protein structure are different as described via the NMR study and are probably caused by disruption of electrostatic interactions.

Figure 5. 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 SDSPAGE.

showed that, upon cross-linking by BS3, the enzyme activity decreased to 80% and 71% at the protein concentration of 0.2 and 1.0 mg/mL, respectively, and then remained at a constant level of 2.0 mg/mL. 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 SDS-PAGE. 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 crosslinkers. 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 toward a stabilized conformation using the molecular crowding effect.49

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02863. Tables of identified cross-links and monolinks upon cross-linking 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 1 H−15N TROSY spectra of unmodified hCA-I, hCA-I cross-linked by 10 molar excess of BS3/BS2G, and hCAI 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

■

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 1111


Article


■

(16) Chen, Z. A.; Jawhari, A.; Fischer, L.; Buchen, C.; Tahir, S.; Kamenski, T.; Rasmussen, M.; Lariviere, L.; Bukowski-Wills, J. C.; Nilges, M.; Cramer, P.; Rappsilber, J. EMBO J. 2010, 29, 717−726. (17) Lauber, M. A.; Reilly, J. P. J. Proteome Res. 2011, 10, 3604− 3616. (18) Leitner, A.; Joachimiak, L. A.; Bracher, A.; Monkemeyer, L.; Walzthoeni, T.; Chen, B.; Pechmann, S.; Holmes, S.; Cong, Y.; Ma, B.; Ludtke, S.; Chiu, W.; Hartl, F. U.; Aebersold, R.; Frydman, J. Structure 2012, 20, 814−825. (19) Kalisman, N.; Adams, C. M.; Levitt, M. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 2884−2889. (20) Bohn, S.; Beck, F.; Sakata, E.; Walzthoeni, T.; Beck, M.; Aebersold, R.; Forster, F.; Baumeister, W.; Nickell, S. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 20992−20997. (21) Kao, A.; Randall, A.; Yang, Y.; Patel, V. R.; Kandur, W.; Guan, S.; Rychnovsky, S. D.; Baldi, P.; Huang, L. Mol. Cell. Proteomics 2012, 11, 1566−1577. (22) Karadzic, I.; Maupin-Furlow, J.; Humbard, M.; Prunetti, L.; Singh, P.; Goodlett, D. R. Proteomics 2012, 12, 1806−1814. (23) Kao, A.; Chiu, C. L.; Vellucci, D.; Yang, Y.; Patel, V. R.; Guan, S.; Randall, A.; Baldi, P.; Rychnovsky, S. D.; Huang, L. Mol. Cell. Proteomics 2011, 10, M110.002212. (24) Lasker, K.; Forster, F.; Bohn, S.; Walzthoeni, T.; Villa, E.; Unverdorben, P.; Beck, F.; Aebersold, R.; Sali, A.; Baumeister, W. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 1380−1387. (25) Jacobsen, R. B.; Sale, K. L.; Ayson, M. J.; Novak, P.; Hong, J.; Lane, P.; Wood, N. L.; Kruppa, G. H.; Young, M. M.; Schoeniger, J. S. Protein Sci. 2006, 15, 1303−1317. (26) Kukacka, Z.; Rosulek, M.; Strohalm, M.; Kavan, D.; Novak, P. Methods 2015, 89, 112−120. (27) Fischer, L.; Chen, Z. A.; Rappsilber, J. J. Proteomics 2013, 88, 120−128. (28) Chen, Z. A.; Fischer, L.; Cox, J.; Rappsilber, J. Mol. Cell. Proteomics 2016, 15, 2769−2778. (29) Brodie, N. I.; Makepeace, K. A.; Petrotchenko, E. V.; Borchers, C. H. J. Proteomics 2015, 118, 12−20. (30) Walzthoeni, T.; Joachimiak, L. A.; Rosenberger, G.; Rost, H. L.; Malmstrom, L.; Leitner, A.; Frydman, J.; Aebersold, R. Nat. Methods 2015, 12, 1185−1190. (31) Brodie, N. I.; Popov, K. I.; Petrotchenko, E. V.; Dokholyan, N. V.; Borchers, C. H. Sci. Adv. 2017, 3, e1700479. (32) Belsom, A.; Schneider, M.; Fischer, L.; Brock, O.; Rappsilber, J. Mol. Cell. Proteomics 2016, 15, 1105−1116. (33) Banerjee, A. L.; Swanson, M.; Mallik, S.; Srivastava, D. K. Protein Expression Purif. 2004, 37, 450−454. (34) Schilling, B.; Row, R. H.; Gibson, B. W.; Guo, X.; Young, M. M. J. Am. Soc. Mass Spectrom. 2003, 14, 834−850. (35) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277−293. (36) Goddard, T. D.; Kneller, D. G. SPARKY 3; University of California: San Francisco, 2008. (37) Merkley, E. D.; Cort, J. R.; Adkins, J. N. J. Struct. Funct. Genomics 2013, 14, 77−90. (38) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 603−614. (39) Sethson, I.; Edlund, U.; Holak, T. A.; Ross, A.; Jonsson, B. H. J. Biomol. NMR 1996, 8, 417−428. (40) Leitner, A.; Walzthoeni, T.; Kahraman, A.; Herzog, F.; Rinner, O.; Beck, M.; Aebersold, R. Mol. Cell. Proteomics 2010, 9, 1634−1649. (41) Kruppa, G. H.; Schoeniger, J.; Young, M. M. Rapid Commun. Mass Spectrom. 2003, 17, 155−162. (42) Swaim, C. L.; Smith, J. B.; Smith, D. L. J. Am. Soc. Mass Spectrom. 2004, 15, 736−749. (43) Leavell, M. D.; Novak, P.; Behrens, C. R.; Schoeniger, J. S.; Kruppa, G. H. J. Am. Soc. Mass Spectrom. 2004, 15, 1604−1611. (44) Onisko, B.; Fernandez, E. G.; Freire, M. L.; Schwarz, A.; Baier, M.; Camina, F.; Garcia, J. R.; Rodriguez-SegadeVillamarin, S.; Requena, J. R. Biochemistry 2005, 44, 10100−10109. (45) Kalkhof, S.; Sinz, A. Anal. Bioanal. Chem. 2008, 392, 305−312.

intensity of unmodified and modified peptides upon cross-linking with BS3 or BS2G determined for the first 7 lysine residues; the monolinks and cross-links identified by mass spectrometry after cross-linking of hCA-I; residue fluctuation (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +420 241 062 156. Tel.: +420 325 873 610 (P.N.). ORCID

Petr Novák: 0000-0001-8688-529X 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.

■

ACKNOWLEDGMENTS This work was supported by the Czech Science Foundation (grant number 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), and 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).

■

REFERENCES

(1) Young, M. M.; Tang, N.; Hempel, J. C.; Oshiro, C. M.; Taylor, E. W.; Kuntz, I. D.; Gibson, B. W.; Dollinger, G. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 5802−5806. (2) Farmer, T. B.; Caprioli, R. M. Biol. Mass Spectrom. 1991, 20, 796−800. (3) Rossi, V.; Gaboriaud, C.; Lacroix, M.; Ulrich, J.; FontecillaCamps, J. C.; Gagnon, J.; Arlaud, G. J. Biochemistry 1995, 34, 7311− 7321. (4) Havel, T. F.; Crippen, G. M.; Kuntz, I. D.; Blaney, J. M. J. Theor. Biol. 1983, 104, 383−400. (5) Havel, T. F.; Kuntz, I. D.; Crippen, G. M. J. Theor. Biol. 1983, 104, 359−381. (6) Rappsilber, J. J. Struct. Biol. 2011, 173, 530−540. (7) Sinz, A.; Wang, K. Biochemistry 2001, 40, 7903−7913. (8) Muller, D. R.; Schindler, P.; Towbin, H.; Wirth, U.; Voshol, H.; Hoving, S.; Steinmetz, M. O. Anal. Chem. 2001, 73, 1927−1934. (9) Wine, R. N.; Dial, J. M.; Tomer, K. B.; Borchers, C. H. Anal. Chem. 2002, 74, 1939−1945. (10) Novak, P.; Young, M. M.; Schoeniger, J. S.; Kruppa, G. H. Eur. Mass Spectrom. 2003, 9, 623−631. (11) Dihazi, G. H.; Sinz, A. Rapid Commun. Mass Spectrom. 2003, 17, 2005−2014. (12) Rozbesky, D.; Man, P.; Kavan, D.; Chmelik, J.; Cerny, J.; Bezouska, K.; Novak, P. Anal. Chem. 2012, 84, 867−870. (13) Rozbesky, D.; Sovova, Z.; Marcoux, J.; Man, P.; Ettrich, R.; Robinson, C. V.; Novak, P. Anal. Chem. 2013, 85, 1597−1604. (14) Kahraman, A.; Herzog, F.; Leitner, A.; Rosenberger, G.; Aebersold, R.; Malmstrom, L. PLoS One 2013, 8, e73411. (15) Jennebach, S.; Herzog, F.; Aebersold, R.; Cramer, P. Nucleic Acids Res. 2012, 40, 5591−5601. 1112


Article

Analytical Chemistry (46) Madler, S.; Bich, C.; Touboul, D.; Zenobi, R. J. Mass Spectrom. 2009, 44, 694−706. (47) Kahraman, A.; Malmstrom, L.; Aebersold, R. Bioinformatics 2011, 27, 2163−2164. (48) Shen, Y.; Delaglio, F.; Cornilescu, G.; Bax, A. J. Biomol. NMR 2009, 44, 213−223. (49) Wang, Y.; Sarkar, M.; Smith, A. E.; Krois, A. S.; Pielak, G. J. J. Am. Chem. Soc. 2012, 134, 16614−16618. (50) Novak, P.; Kruppa, G. H.; Young, M. M.; Schoeniger, J. J. Mass Spectrom. 2004, 39, 322−328. (51) Guo, X.; Bandyopadhyay, P.; Schilling, B.; Young, M. M.; Fujii, N.; Aynechi, T.; Guy, R. K.; Kuntz, I. D.; Gibson, B. W. Anal. Chem. 2008, 80, 951−960.

1113


Impact of Chemical Cross-Linking on Protein Structure and Function

Recommend Documents