Article pubs.acs.org/Langmuir
Effects of Urea on Selectivity and Protein−Ligand Interactions in Multimodal Cation Exchange Chromatography Melissa A. Holstein,†,§ Siddharth Parimal,†,§ Scott A. McCallum,‡,§ and Steven M. Cramer*,†,§ †
Howard P. Isermann Department of Chemical and Biological Engineering and ‡Department of Biology, §Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States S Supporting Information *
ABSTRACT: Nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were employed in concert with chromatography to provide insight into the effect of urea on protein−ligand interactions in multimodal (MM) chromatography. Chromatographic experiments with a protein library in ion exchange (IEX) and MM systems indicated that, while urea had a significant effect on protein retention and selectivity for a range of proteins in MM systems, the effects were much less pronounced in IEX. NMR titration experiments carried out with a multimodal ligand, and isotopically enriched human ubiquitin indicated that, while the ligand binding face of ubiquitin remained largely intact in the presence of urea, the strength of binding was decreased. MD simulations were carried out to provide further insight into the effect of urea on MM ligand binding. These results indicated that, while the overall ligand binding face of ubiquitin remained the same, there was a reduction in the occupancy of the MM ligand interaction region along with subtle changes in the residues involved in these interactions. This work demonstrates the effectiveness of urea in enhancing selectivity in MM chromatographic systems and also provides an in-depth analysis of how MM ligand−protein interactions are altered in the presence of this fluid phase modifier.
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INTRODUCTION Efficient bioseparation processes are crucial for the production of high-purity pharmaceuticals and therapeutics. In addition, high-resolution separations are becoming increasingly important for complex bioanalytical applications. Recent advances in the design of multimodal (MM) chromatographic systems have produced new classes of chromatographic materials which can provide alternative and improved affinities and selectivities as compared to traditional single-mode chromatographic materials such as ion exchangers.1−8 MM chromatographic materials have been used to improve the selectivity of biomolecules such as peptides,2 oligonucleotides,9 nucleic acids,10,11 and oligomer-like compounds.12 Furthermore, MM resins have been shown to enhance chromatographic separations, to improve product quality, and to improve process efficiency in industrial-scale manufacturing processes.13,14 Numerous studies have examined different stationary phase characteristics of MM resins including the effects of ligand density, spacer arm length, and the substitution of different functional groups.15−19 We have previously investigated the underlying nature of protein binding in ion exchange (IEX) and MM chromatographic systems using NMR and coarse-grained ligand docking simulations in concert with chromatography data for a library of ubiquitin mutants.20 Specific protein−ligand interaction sites were identified by NMR and ranked based on their binding affinity. Interestingly, the binding sites determined in that study clustered to form a © 2012 American Chemical Society
preferred binding region on the ubiquitin protein surface. Coarse-grained ligand docking simulations were then employed to study the modes of interaction between the MM ligand and the identified preferred binding region. There is a large body of literature examining the effects of mobile phase modifiers on protein retention behavior in traditional modes of chromatography.21−25 There have also been a few recent papers on the effects of mobile phase modifiers in MM chromatography. Gao et al. investigated the effects of mobile phase conditions, pH, and salt concentration on the adsorption behavior of bovine serum albumin on a MM cation exchange adsorbent developed for expanded bed adsorption.26,27 We have recently carried out detailed studies into the effects of different mobile phase additives on protein adsorption and selectivity in MM chromatography.28−30 Interactions of urea with biological macromolecules such as proteins have been studied at length through various experimental and theoretical approaches.31−37 Recent experimental NMR work has combined statistical coil ensemble modeling and small-angle scattering to analyze the conformational behavior of ubiquitin in the presence of urea.38 While these studies have concentrated primarily on examining protein unfolding, the mechanism by which urea affects protein Received: June 11, 2012 Revised: November 30, 2012 Published: November 30, 2012 158
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Table 1. Ligand Structures and Total Ionic Capacities of a Strong Cation Exchanger (SP Sepharose FF), a Weak Cation Exchanger (CM Sepharose FF), and a Multimodal Cation Exchanger (Capto MMC)a
a
A portion of the multimodal ligand is outlined by a box, which denotes the representative multimodal ligand (N-benzoyl-DL-methionine) used in the nuclear magnetic resonance experiments. MA). Sodium hydroxide was purchased from Thermo Fisher Scientific (Pittsburgh, PA). Equipment. Analytical linear gradient experiments were carried out using a Waters HPLC system consisting of a 600 multisolvent delivery system, a 717 WISP autoinjector, and a 996 photodiode array detector controlled by a Millennium chromatography software manager. NMR spectra were obtained at 25 °C using a Bruker 600 MHz spectrometer equipped with a 1H/13C/15N cryoprobe with z-axis gradients. Procedures. Linear Gradient Chromatography. Linear gradient chromatography experiments were carried out at room temperature on cation exchange (SP Sepharose FF and CM Sepharose FF) and MM cation exchange (Capto MMC) resins in the absence and presence of 2 M urea. The resin structures and ionic capacities are given in Table 1. The linear salt gradient transitioned from buffer A (20 mM sodium acetate, pH 5) to buffer B (20 mM sodium acetate, 1.5 M sodium chloride, pH 5) over 45 column volumes. The experiments with urea consisted of these buffer conditions with the addition of 2 M urea in both buffers. Linear gradient experiments were also performed on the MM column at pH 7 in the absence and presence of 2 M urea. For these experiments, the salt gradient transitioned from buffer A (20 mM sodium phosphate, pH 7) to buffer B (20 mM sodium phosphate, 1.5 M sodium chloride, pH 7). A flow rate of 1 mL/min and a column volume of 1 mL were used in all experiments. The column effluent was monitored using UV absorbance at 280 nm. Protein Expression and Purification. Uniformly 13C/15N-enriched wild-type recombinant ubiquitin was expressed using the BL21(DE3) strain of E. coli and purified as described by Chung et al.20 The final protein concentration was determined by spectrophotometric analysis at 280 nm with a molar extinction coefficient of 1490 M−1 cm−1.44 NMR. NMR data were acquired and processed using Bruker TopSpin 2.1 software and analyzed using the software package Sparky.45 Assignments of amide resonances were confirmed by matching published chemical shift values (BMRB accession number 6457) with backbone correlation patterns detected in spectra of unbound protein. 1H−15N HSQC spectra were acquired where each sample had an initial volume of 320 μL and contained 0.1 mM isotopically enriched ubiquitin in NMR buffer (10 mM sodium acetate, pH 5.0, 0.02% sodium azide, 1 μM TMSP as an internal standard, and 5% D2O) with protein alone and in the presence of of 0.5 M, 1.0 M, 1.5 M, and 2.0 M urea. Titrations were performed where spectra were acquired of the protein over a range of MM ligand (N-benzoyl-DLmethionine) concentrations in the absence and presence of 2 M urea. This model soluble ligand is structurally equivalent to the functional
interactions by either directly associating with protein atoms or by altering water structure is less well understood.39−43 This lack of mechanistic understanding of protein−urea interactions at low concentrations of urea has made it difficult to explain and predict the effects of urea in chromatographic processes, especially ones which operate through multiple modes of interaction. In this investigation, experimental and theoretical techniques are employed to provide insight into changes in protein adsorption and selectivity in the presence of urea. IEX and MM chromatography are first used to examine the differences in retention behavior of a diverse protein library in the presence of urea. NMR titration experiments are then carried out in the presence of urea to determine the effect of this modifier on sitespecific protein−ligand binding for the protein ubiquitin. MD simulations are then carried out to provide further insight into the effect of urea on MM ligand binding. This work demonstrates the effectiveness of urea in enhancing selectivity in MM chromatographic systems and also provides an in-depth analysis of how MM ligand−protein interactions are altered in the presence of this fluid phase modifier.
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EXPERIMENTAL SECTION
Materials. SP Sepharose FF, CM Sepharose FF, and Capto MMC media were obtained from GE Healthcare (Uppsala, Sweden) and packed into separate Pharmacia Biotech glass columns (5 mm × 50 mm). Urea, hydrochloric acid, sodium chloride, acetic acid, sodium acetate, deuterium oxide (D2O), 3-(trimethylsilyl)propionic acid-d4 sodium salt (TMSP), NMR tubes, N-benzoyl-DL-methionine, sodium phosphate (monobasic and dibasic), lectin (peanut), albumin (bovine, human, pig, and sheep), conalbumin (chicken egg white), trypsinogen (bovine pancreas), β-lactoglobulin A (bovine milk), β-lactoglobulin B (bovine milk), trypsin (bovine and porcine), lipoxidase (soybean), αchymotrypsin (bovine pancreas), α-chymotrypsinogen A (bovine pancreas), β-chymotrypsin (bovine pancreas), ribonuclease B (bovine pancreas), cytochrome c (horse heart), ribonuclease A (bovine pancreas), L-glutamic dehydrogenase (bovine liver), aprotinin (bovine lung), lysozyme (chicken egg white), papain (papaya latex), and avidin (egg white) were purchased from Sigma-Aldrich (St. Louis, MO). Ubiquitin (human) was purchased from Boston Biochem (Cambridge, 159
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Table 2. List of Properties of the Proteins Used in Chromatography Experimentsa formal charge (pH = 7)
ASA (Å2)
FASA+ (pH = 5)
FA5A− (pH = 5)
PDB ID
peanut lectin
2PEL
24.7
5.8
−5.00
−5.00
9872.25
0.64
0.36
bovine serum albumin
3V03
66.3
5.3
−7.00
−13.00
28228.61
O.63
0.3/
1UBQ
8.6
6.8
1.00
0.00
4675.96
0.64
0.36
lAO6
66.4
5.2
0.00
−5.00
27529.75
0.64
0.36
trypsinogen
1TGB
23.3
9.3
8.00
6.00
8859.07
0.68
0.32
β-lactoglobulin A
1B0O
18.2
5.1
−6.00
−6.00
8412.18
0.64
0.36
β-lactoglobulin B
1BSQ
18.3
5.3
−6.00
−6.00
7884.47
0.64
0.36
pig albumin bovine trypsin
N/A 1S0Q
N/A 23.8
4.8−5 10.3
N/A 7.00
N/A 7.00
N/A 8980.63
N/A 0.66
N/A 0.32
Lipoxidase
1F8N
94.4
5.65
5.00
−3.00
31361.57
0.66
0.34
α-chymotrypsin β-chymotrypsin α-chymotrypsinogen A
5CHA N/A 2CGA
25 N/A 25.7
9.17 5.2 8.97
3.00 N/A 6.00
3.00 N/A 5.00
10718.37 N/A 10120.07
0.67 N/A 0.67
0.33 N/A 0.33
RNaseB
1RBB
13.7
9.74
7.00
4.00
6652.15
0.69
0.31
bovine cytochrome c
2B4Z
11.6
10.25
8.00
8.00
5849.52
0.71
0.29
RNaseA
1RBX
13.7
9.6
7.00
6.00
6581.81
0.70
0.30
horse cytochrome c
1HRC
11.7
10.25
9.00
8.00
5793.34
0.71
0.29
aprotinin
1PIT
6.5
10.5
6.00
6.00
3905.23
0.72
0.28
lysozyme
1AKI
14.3
11.35
9.00
9.00
6430.87
0.70
0.30
1VYO
28.7
10
14.00
13.00
15655.26
0.68
0.32
protein
human ubiquitin human serum albumin
avidin a
formal charge (pH = 5)
size (kDa)
Pl
FA5H (pH = 5)
structural information
0.53 4 domains, mainly beta (4% helical, 48% beta) 0.60 3 structurally similar domains, 2 subdomains in each (74% helical content) 0.49 1 domain (23% helical, 34% β sheet) 0.56 3 structurally similar domains, 2 subdomains in each (70% helical content) 0.54 1 domain, mainly beta (10% helical, 34% beta) 0.59 1 domain, mainly beta (13% helical, 39% betaj 0.55 1 domain, mainly beta (16% helical, 37% beta) N/A N/A 0.51 1 domain, mainly beta (10% helical, 34% beta) 0.56 2 structural domains (41% helical, 14% beta) 0.51 1 domain, 3 chains, mainly beta N/A N/A 0.54 2 domains, mainly beta (14% helical, 35% beta and 12% helical-35%beta) 0.57 1 domain, alpha-beta (20% helical, 35% beta) 0.56 1 domain, mainly alpha (40% helical, 1% beta) 0.56 1 domain, alpha-beta (20% helical, 35% beta) 0.56 1 domain, mainly alpha (40% helical, 1% beta) 0.60 1 domain, irregular (18% helical, 25% beta) 0.47 1 domain, mainly alpha (41% helical, 10% beta) 0.53 1 domain, mainly beta (7% helical, 46% beta)
ASA = Accessible surface area, FASA+ = Fractional positive ASA, FASA− = Fractional negative ASA, FASH = Fractional hydrophobic ASA.
moieties of the immobilized resin material (indicated by a box in Table 1) with the exception of a modification at the resin linkage point. Due to the limited solubility of the ligand, the maximum ligand concentration was limited to 3.2 mM. The maximum change in weighted average chemical shift (ΔδNH = [(ΔδH)2 + (0.2 · ΔδN)2]1/2) was calculated for each amide group detected free of spectral overlap in the NMR experiment that was detected to undergo ligand-induced changes in chemical shift.46,47 Curve fitting and calculations were performed with Origin 8 (OriginLab) and protein visualization was carried out with PyMol.48 Molecular Dynamics Simulations. The initial structure of human ubiquitin was obtained from the RCSB Protein Data Bank (PDB ID: 1D3Z). The crystal structure was protonated and the ionizable residues were assigned protonation states at a pH of 5 (similar to chromatography experiments). At this pH, lysines, arginines, and histidines were positively charged, whereas glutamic acids and aspartic acids were negatively charged, resulting in an overall charge of +1e for ubiquitin. The force field used for the protein was AMBER 94.49 For the ligands, parameters were taken from previous work by our group.50 MD simulations were performed for the system consisting of one protein molecule and six MM ligand molecules per simulation box with explicit SPC/E51 water molecules and counterions using the GROMACS 3.3.3 molecular dynamics package.52,53 To minimize the effect of starting orientations, the spatial arrangement of the six ligand
molecules around the protein was chosen at random, and the ligands were placed sufficiently far away (>2 nm) from the protein molecule. This procedure was repeated six times to obtain six different starting configurations of the system for MD simulations. Each of the six configurations was simulated for 40 ns, providing data for a total of 240 ns. To understand the effect of urea on MM ligand−protein interactions, MD simulations were performed with similar systems in the presence of 380 urea molecules (approximately 2 M) in each of the simulation boxes. The Kirkwoord-Buff model was used to parametrize the atoms in the urea molecule and their interactions with other molecules in the system.54 Each system was initially energy-minimized using the steepest descent algorithm before performing MD simulations in the isothermal−isobaric (N,P,T) ensemble. Temperature (300 K) and pressure (1 bar) were maintained using the Nose-Hoover thermostat55,56 and Parrinello-Rahman barostat,57 respectively. Periodic boundary conditions were applied in all three directions. A cutoff of 1 nm was used for all LJ interactions. The Particle Mesh Ewald (PME) method58 was used to calculate electrostatic interactions. The software packages VMD59 and PyMol48 were used for visualization and simulation setup. 160
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Figure 1. Chromatographic retention data under linear salt gradient elution conditions for a protein library on different resins in the absence (black) and presence (gray) of 2 M urea. (a) Strong cation exchanger (SP Sepharose FF) results at pH 5, (b) Weak cation exchanger (CM Sepharose FF) results at pH 5, (c) Multimodal cation exchanger (Capto MMC) results at pH 5, (d) Multimodal cation exchanger (Capto MMC) results at pH 7. Proteins that did not elute under the linear gradient are denoted by *.
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RESULTS AND DISCUSSION Chromatographic Retention Behavior. A library of commercially available proteins was employed to study the effect of urea on protein chromatographic behavior. The proteins were selected to sample a range of chemical and physical properties such as hydrophobicity, charge, and size (Table 2). Chromatographic retention times were determined for each protein in the absence and presence of urea on strong cation exchange (SP Sepharose FF), weak cation exchange (CM Sepharose FF), and MM cation exchange (Capto MMC) chromatographic resins. The structures and ionic capacities of these resins are shown in Table 1. As seen in the table, the Capto MMC ligand includes electrostatic, hydrogen bonding, aromatic, and thiophilic interaction moieties. As described in the Experimental Section, linear gradient experiments were carried out with the ion exchange resins at pH 5 and the MMC resin at pH 5 and 7. Representative chromatograms for horse cytochrome c and peanut lectin are presented in Figure S1. Figure 1 displays the salt concentration required to elute each protein from the three chromatographic resins in the absence and presence of urea. As can be seen in Figure 1a,b, nearly all of the proteins experienced a slight decrease of retention in the cation exchangers in the presence
of urea. The most likely explanation of this general trend is the reduced electrostatic interactions that occur due to the elevated dielectric constant in the presence of urea (note: at 2 M urea, the dielectric constant increases by roughly 7%, ref 60). The decrease in retention of these proteins could also be partly due to a reduction in hydrogen bonding that can occur in the presence of urea.42,61 It is interesting to note that some proteins (BSA on SP; BSA, HSA, and pig albumin on CM) exhibited a slight increase in retention in the presence of urea. In contrast to the cation exchange results, the proteins exhibited markedly different responses to urea on the MM resin at pH 5 (Figure 1c). While almost all of the proteins exhibited a decrease in retention in the presence of urea, some of them exhibited significant decreases leading to shifts in selectivity. This resulted in a significant alteration of the elution order in the presence of urea. Since urea is known to weaken hydrophobic interactions and the proteins possessed various degrees of hydrophobicity, it was expected that they would have different responses to urea. However, upon examining these retention results along with the protein property data presented in Table 2, it can be seen that there were no clear trends in a protein’s response to urea and the protein’s fractional hydrophobic surface area, or any single protein property. For 161
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Figure 2. Multimodal (MM) ligand binding sites identified by NMR with human ubiquitin in the absence (a) and presence (b) of 2 M urea (data for (a) are taken from ref 20). Residues are colored on a red−white−blue scale based on their apparent dissociation constants (KD) where red indicates high binding strength (KD ≈ 0.01 mM) and blue indicates low binding strength (KD ≥ 5 mM). Residues that displayed multiphasic behavior are depicted in gray. A yellow dashed line denotes the “sub-region” which exhibited a significant decrease in binding strength in the presence of urea.
example, α-chymotrypsinogen A and horse cytochrome c have very similar hydrophobic surface areas. However, while αchymotrypsinogen A and horse cytochrome c eluted at nearly identical salt concentrations in the absence of urea, very different salt concentrations were required to elute these proteins when urea was present in the mobile phase. In fact, this occurred with a number of protein pairs. We believe that this is due to the complex nature of the multiple interactions that occur with MM ligands as well as the importance of the distribution of protein properties on the protein surfaces. By comparing the elution salt concentrations in Figure 1a−c, it can be seen that higher salt concentrations were required to elute proteins from the MM resin than from the cation exchange resins under identical experimental conditions. This indicates that the combination of interactions greatly enhanced protein adsorption to the MM resin, which is consistent with previous results.20,30 In fact, several of the proteins (i.e., lysozyme, aprotinin, and avidin) were not eluted from the MM resin during the 1.5 M salt gradient under pH 5 buffer conditions. In order to obtain quantitative retention data for the strongly bound proteins on the MM resin, experiments were also performed at a higher pH value where the proteins were less strongly bound. Figure 1d presents the salt concentrations required to elute the proteins under pH 7 gradient conditions. As can be seen in the figure, retention data were obtained for all of the proteins, including those that did not elute at pH 5. It is interesting to note that many of the proteins in this library have pI values below 7 (Table 2), which indicates that they had a net negative charge during this gradient experiment. The fact that they were still binding to the column at these conditions implies that hydrophobic interactions and hydrogen bonding were able to overcome any electrostatic repulsion that may have occurred between the negatively charged proteins and the cation exchange surface. In addition, the distribution of charges on these proteins could also have played a role as has been recognized for many years starting with the seminal work of Regnier and co-worker as well as many others.62−67
These chromatographic results demonstrated that protein binding affinity and selectivity with urea were significantly different for the cation exchange and MM chromatographic systems. Urea proved to be very effective in enhancing and creating windows of selectivity among the proteins in this library. In order to gain further insight into the protein−ligand binding interactions and how they were affected by the addition of urea, NMR and MD simulations were employed as described in the following sections. 1 H−15N HSQC NMR Titration Results. In the chromatography experiments, the protein ubiquitin exhibited a slight decrease in retention due to urea on the cation exchange resin while exhibiting a significant decrease in retention on the MM resin. To further examine this behavior, NMR titration experiments were carried out using isotopically enriched ubiquitin as a model protein. Protein−ligand interactions in solution were monitored by a series of 1H−15N HSQC spectra at varying ligand concentrations. Two-dimensional 1H−15N HSQC spectra were first acquired with protein alone (no ligand or urea) and with several concentrations of urea (0.5 M, 1 M, 1.5 M, 2 M). The resulting spectra at these urea concentrations showed no significant chemical shift perturbations indicating that there was no protein unfolding or association of the urea with the protein surface. Combined chemical shifts at 2.0 M urea (in the absence of ligand) are plotted for all residues in Figure S2. The titration with the MM ligand was then carried out in the presence of 2 M urea to examine the effect of urea on protein− ligand interactions in solution. Labeled amide groups on the protein that came into close proximity and interacted with the ligand experienced a change in chemical shift due to perturbations in the local electronic environment. These amide groups served as reporters of the local electronic environment for each residue. A single resonance peak for the unbound and ligand bound forms of the protein was observed for each amide group in the series of two-dimensional 1H−15N HSQC spectra. Representative 1H−15N HSQC spectra are presented in Figures S3−S4. The line widths were largely free of exchange broadening and had ligand-dependent chemical 162
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Figure 3. Molecular dynamics simulation results for a multimodal (MM) ligand binding to ubiquitin in the absence and presence of urea. (a) Chemical structure of the MM ligand used in the MD simulations denoting the four possible modes of interaction with the protein. (b,c) MD simulation results for MM ligand−protein interactions in the absence and presence of urea, respectively. The ubiquitin molecule is colored on a red− white−blue scale based on the ligand proximity count for each protein atom where red indicates the maximum probability of interactions (approximately 56%) and blue indicates no interactions. The regions outlined in yellow exhibited a change in MM ligand−protein interaction upon the addition of urea. While regions I and II showed a decrease in interactions, region III exhibited an increase in interactions.
shift values characteristic of “fast exchange” behavior for protein−ligand interactions. As a result, the observed resonance frequencies were assumed to be time-averaged and were fit with a single-site binding model as described previously20 to determine apparent dissociation constants (KD). The observed changes in chemical shift indicated that there were some residues whose perturbations were induced by ligand binding to a single proximal site and others that displayed more complex multisite or multiphasic behavior, which occurs when a spin experiences chemical shift perturbations due to multiple noncompeting binding events. Dissociation constants for MM ligand binding in the presence of urea were determined for each residue of ubiquitin. The results are shown in Figure 2 where the dissociation constants in the presence of urea are compared with previously determined values from ligand−protein titration experiments in the absence of urea20 (note: only one face of the protein is presented in the figure since all residues with KD values less than 5 mM occurred on this face). Figure 2 is based on a red− white−blue color scale where red indicates a KD value of 0.01 mM and blue indicates a KD value of 5 mM or more. Residues which exhibited multiphasic behavior are colored in gray in the figure. A comparison of Figure 2a,b reveals the effect of urea on MM ligand binding as evaluated by NMR. The overall effect of urea was a decrease in binding affinity to the MM ligand, consistent with the chromatography results where the elution salt decreased from 1.24 to 0.88 M. The NMR data also provided information about the residues that were responsible for this decrease. As can be seen in the figure, the binding face of ubiquitin remained essentially the same in the presence and absence of urea. Interestingly, while some residues (G10, R42, I44) showed minimal changes in dissociation constants upon the addition of urea, other residues (A46, G47, Y59, and T66) exhibited significant decreases in binding strength. In fact, the residues that exhibited a marked decrease were all located in a specific “sub-region” in the binding face as indicated in the figure. This suggests that urea may have a selective behavior in
affecting MM interactions on specific regions on the protein surface as opposed to an overall weakening of intermolecular interactions. It is also interesting to note that, in the presence of urea, the MM ligand binding to residues L73 and R72 was increased. Further, residues L8 and V70 displayed simple two-state binding behavior, in contrast to the multiphasic behavior exhibited by these two residues in the absence of urea (Figure S5). There are two possible explanations for this observed change in behavior. First, the two binding sites which included these residues may have become similar in their binding strength, resulting in a linear spin trajectory. Second, one of the binding sites comprising these residues could be weakened to such an extent in the presence of urea that the binding induced changes in chemical shift to these residues is now dominated by a single binding site. In order to obtain further insight into MM ligand−protein binding in the presence and absence of urea, MD simulations were carried out as described in the next section. MD Simulation Results. All-atom MD simulations were carried out to examine the effect of urea on the binding interactions between ubiquitin and the MM ligand in free solution. To identify ligand binding regions on the protein surface, an analysis was carried out similar to what has been presented previously.50 The MM ligand was divided into four possible interaction moieties (Figure 3a) and the distance of each moiety from protein atoms was observed throughout the course of the simulation. The ligand was considered to be “bound” to the protein at a given time if the center of masses of at least three of the four interaction moieties were within 5.5 Å of the protein surface. All of the protein atoms within 4.5 Å of any atom of a “bound” ligand were then considered to be interacting with the ligand. These proximity counts for each protein atom were averaged over time and the results are mapped on the protein surface as shown in Figure 3b,c. A red− white−blue color scale is used with red representing the highest frequency of interaction and blue representing the lowest frequency. 163
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Comparison of Figure 3b and c reveals that, while the overall binding face of ubiquitin remained the same, there was a reduction in the area of the MM ligand interaction region (based on the atom proximity counts) along with subtle changes in the residues which participated in these interactions. Three separate regions could be identified on the binding face of ubiquitin, which were affected by urea. While regions I and II showed a decrease in ligand interactions, region III showed an increase in the ligand counts during the MD simulations. An examination of the positive electrostatic potential (EP) on this face of the protein indicates that these three regions all exhibit positive EP.50,68 As described above, it is expected that the elevated dielectric constant in the presence of urea would result in decreased electrostatic interactions. While this explains the behavior of regions I and II, it does not explain the increased interactions observed for region III in the MD simulations. The hydropathy indices of the residues on this face of the protein indicate that regions I and III have significant surface hydrophobicity while region II does not.50 Clearly, an analysis of the surface EP and hydrophobicity of the protein do not explain the regionally selective effect of urea observed in the MD simulations. However, recent work69 has shown that hydrophobic indices of amino acids are not the most ideal indicators of surface hydrophobicity on a protein’s surface. Therefore, in order to shed light on this “regionally selective” behavior, future work will combine better hydrophobic characterization of ubiquitin’s surface and combine it with free energy calculations in regions I, II, and III to probe the competitive binding of a MM ligand, urea molecules, and water to these regions in order to provide deeper mechanistic insights into urea’s effect based on protein surface properties. The discussion that follows concentrates on the extent of comparisons that can be drawn between MD results and those obtained from NMR experiments. The results obtained from the MD simulations are in general agreement with those obtained from the NMR experiments. While the inherent differences underlying the two techniques make quantitative comparisons challenging, qualitative comparisons can be carried out. NMR detects the changes in the local electronic environment of reporter atoms (in this case, the amide nitrogen of each residue) which can also propagate within the protein through complex mechanisms involving secondary structural elements that may not be directly involved in the MM ligand interactions.70−75 On the other hand, the analysis of MD simulations is based on the proximity of ligands to a protein surface. The MD results (Figure 3b,c) indicated that ligand interactions with the surface-exposed atoms of residue H68 (region II) were decreased significantly in the presence of urea. It turns out that the amide nitrogen of H68 is in close proximity to the urea affected residues in the NMR experiments (Figure 2). This can be clearly observed in Figure 4, where the spatial arrangement of the amide reporter atoms for each residue are presented revealing that the amide nitrogen atoms of H68 and the NMR identified “sub-region” (which includes F45, A46, G47, and K48) are close to each other. This suggests that changes in ligand interactions with the side chain of residue H68 could be responsible for the observed NMR chemical shifts in the “urea affected region” due to the indirect chemical shift propagation in neighboring secondary structural elements. This may explain why the observed “regionally selective” behavior of urea was revealed in MD simulations as an effective decrease in the ligand interaction area while the NMR experiments captured this behavior as a decrease in the
Figure 4. Ribbon representation of ubiquitin with the position of amide nitrogen reporter atoms shown as small spheres. The ribbon and spheres are colored according to a red−white−blue scale based on their apparent dissociation constants (KD) obtained for multimodal ligand−protein binding in the absence of urea. Residues K6, F45, A46, G47, K48, T66, and H68 have been labeled in the figure to illustrate their relative spatial arrangement.
strengths of these protein−ligand interactions. Further, the observed increase in binding affinity for residues L73 and R72 as detected by NMR may be due to the relative flexibility of this top region of the protein which would enable these residues to come into closer proximity to MM ligands interacting with region III identified from the MD simulations (note: region III had higher occupancy in the presence of urea as determined by MD). As stated above, the MD simulations indicated a reduction in the area of the ligand interaction region on the binding face for ubiquitin in the presence of urea. In a solid-phase chromatographic system, this could result in a reduction in the number of MM ligands binding to the protein in the adsorbed state. This reduction in the number of ligands interacting with the protein at a given time would, in turn, likely reduce the overall binding affinity of the protein to the resin in the presence of urea, which is what was observed in the chromatography experiments.
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CONCLUSIONS Chromatography, NMR, and MD simulations were employed to study the nature of protein adsorption and the effects of urea in MM chromatographic systems. The chromatography results with a protein library indicated that urea had a significant effect on protein−ligand binding interactions in MM chromatographic systems. While most of the proteins experienced a slight reduction in retention due to urea in the cation exchange resins, there were significant differences in retention on the MM cation exchanger, giving rise to unique selectivities and reversals in the protein elution order. The lack of correlations 164
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between the effects of urea on a protein’s retention behavior and overall protein properties suggested a protein-selective behavior of urea. In order to gain further insight into the effect of urea, NMR and MD simulations were employed to probe MM ligand binding to ubiquitin. Both of these techniques indicated that urea modulates MM ligand−protein interactions in a “regionally selective” way with different aspects of this behavior being revealed by the two complementary techniques. NMR experiments revealed that, while the binding face of ubiquitin remained intact, the observed dissociation constants for different residues were altered to varying extents in the presence of urea. Results from MD simulations indicated that, while the overall probability of MM interactions on the binding face of ubiquitin was reduced, there were subtle variations in these probabilities across the different residues which could not be fully explained based solely on electrostatic potential or hydropathy indices. This work demonstrates the effectiveness of urea in enhancing selectivity in MM chromatographic systems. While NMR and MD results were consistent with each other and highlighted the “regionally selective” effect of urea, future work should concentrate on investigating the underlying mechanism of this behavior. Further, while the NMR and MD results presented here have focused on ligand−protein binding in solution, it will also be important in the future to examine protein binding to solid resin systems containing the MM ligand in order to elucidate avidity and steric effects that will certainly play a role in these systems.
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ASSOCIATED CONTENT
* Supporting Information S
Representative chromatograms for horse cytochrome c and peanut lectin, combined chemical shifts for all ubiquitin residues in the presence of 2 M urea, 1H−15N HSQC spectra for ubiquitin residues binding to MM ligand in the presence and absence of 2 M urea, 1H−15N HSQC spectra for Leu8 of ubiquitin highlighting multiphasic behavior. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: (518) 894-6198. Fax: (518) 276-4030. E-mail: crames@ rpi.edu. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Foundation grant (CBET 0933169). We also thank George Makhatadze, Mayank Patel, and Werner Streicher for their assistance with the expression of the isotopically enriched ubiquitin.
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