Anal. Chem. 2005, 77, 2157-2165
Electrostatic Contributions to Protein Retention in Ion-Exchange Chromatography. 2. Proteins with Various Degrees of Structural Differences Yan Yao and Abraham M. Lenhoff*
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716
The relation of protein structure to retention provides a framework within which to investigate chromatographic adsorption mechanisms. Protein sets with varying degrees of structural differences were studied to relate variations in protein properties to retention behavior. To explore molecular contributions to protein adsorption in ionexchange chromatography, protein-adsorbent electrostatic interactions were modeled using a continuum approach. The calculations qualitatively capture the chromatographic differentiation of closely related subtilisin variants. Descriptions of the electrostatic interactions of FGF-1 vs FGF-2 with cation exchangers were obtained, and aid in rationalizing differences in experimental retention trends across a set of adsorbents based on different adsorption mechanisms linked to the adsorbent structure. Comparative calculations for proteins with differences in local or overall arginine-lysine composition, including subtilisin variants G166R/G166K and lysozyme/cytochrome c, suggest that continuum electrostatics is not adequate to capture the full quantitative characteristics of the chromatographic retention of proteins. To allow more accurate description of retention, additional molecular interactions, specifically hydration effects, must be incorporated in the model. The coupling of protein physicochemical characteristics and adsorbent properties determines protein retention in chromatographic processes.1 Protein properties such as charge distribution and molecular geometry can significantly influence retention behavior in ion-exchange chromatography (IEC), the effects of which are taken into account in protein purification by manipulating solution conditions as well as choosing adsorbents. Molecular modeling of protein-adsorbent interactions, suitably combined with experimental studies of chromatographic retention, can provide useful information on protein adsorption mechanisms and help inform decisions on design and operating conditions in practice. There have been a large number of modeling studies of protein-surface interactions,2-20 among which those considering * Corresponding author. Fax: +1-302-831-4466. E-mail:
[email protected]. (1) DePhillips, P.; Lenhoff, A. M. J. Chromatogr., A 2001, 933, 57-72. (2) Boardman, N. K.; Partridge, S. M. Biochem. J. 1955, 59, 543-552. (3) Kopaciewicz, W.; Rounds, M. A.; Fausnaugh, J.; Regnier, F. E. J. Chromatogr. 1983, 266, 3-21. (4) Melander, W. R.; ElRassi, Z.; Horva´th, C. J. Chromatogr. 1989, 469, 3-27. 10.1021/ac048733f CCC: $30.25 Published on Web 03/05/2005
© 2005 American Chemical Society
the full 3D structure of proteins and peptides5,7-15,17,18 have provided detailed representations of the underlying molecular events. However, since these models can all typically accommodate adjustable parameters, and since all generally capture attractive interactions in the presence of oppositely charged moieties, comparisons of the model predictions with experimental data are seldom definitive in establishing model validity. Such model discrimination can be aided by comparing the retention of proteins with specific local structural variations to help determine relevant protein structural properties. Measurable retention differences among cytochrome c variants with a maximum of six sequence changes and similar net charge at the experimental pH have been observed on cation exchangers,18,21 highlighting the importance of individual residues in determining the overall adsorption behavior. In a companion paper,18 we showed that continuum electrostatic modeling of protein-adsorbent interactions in IEC can capture these subtle retention changes among cytochrome c variants. Further investigation of the structure-retention relation of proteins with varying degrees of structural dissimilarities may help in evaluating the effectiveness of the simplified electrostatic approach in modeling adsorption, as well as probing the relative importance of molecular interactions in determining macroscopic retention behavior. Here we study three additional protein families in further exploring, with the aid of electrostatic computations, (5) Lu, D. R.; Park, K. J. Biomater. Sci., Polym. Ed. 1990, 1, 243-260. (6) Ståhlberg, J.; Jo ¨nsson, B.; Horva´th, C. Anal. Chem. 1991, 63, 1867-1874. (7) Yoon, B. J.; Lenhoff, A. M. J. Phys. Chem. 1992, 96, 3130-3134. (8) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962-972. (9) Roush, D. J.; Gill, D. S.; Willson, R. C. Biophys. J. 1994, 66, 1290-1300. (10) Noinville, V.; Vidal-Madjar, C.; Se´bille, B. J. Phys. Chem. 1995, 99, 15161522. (11) Juffer, A. H.; Argos, P.; De Vlieg, J. J. Comput. Chem. 1996, 17, 17831803. (12) Ben-Tal, N.; Honig, B.; Peitzsch, R. M.; Denisov, G.; McLaughlin, S. Biophys. J. 1996, 71, 561-575. (13) Tobias, D. J.; Mar, W.; Blasie, J. K.; Klein, M. L. Biophys. J. 1996, 71, 29332941. (14) Asthagiri, D.; Lenhoff, A. M. Langmuir 1997, 13, 6761-6768. (15) Ravichandran, S.; Madura, J. D.; Talbot, J. J. Phys. Chem. B 2001, 105, 3610-3613. (16) Latour, R. A.; Hench, L. L. Biomaterials 2002, 23, 4633-4648. (17) Zhou, J.; Tsao, H. K.; Sheng, Y. J.; Jiang, S. Y. J. Chem. Phys. 2004, 121, 1050-1057. (18) Yao, Y.; Lenhoff, A. M. Anal. Chem. 2004, 76, 6743-6752. (19) Hlady, V.; Buijs, J. Curr. Opin. Biotechnol. 1996, 7, 72-77. (20) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110-115. (21) Kundu, A.; Barnthouse, K. A.; Cramer, S. M. Biotechnol. Bioeng. 1997, 56, 119-129.
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005 2157
the influence of local molecular properties on protein affinity to ion exchangers. In each case, we compare computational results with previously reported experimental data from several different groups for systems of proteins differing to varying degrees in primary sequence. Such an approach provides a frame of reference that allows qualitative and quantitative trends to be analyzed. The first system is a set of subtilisin mutants with substitutions, including of ionizable and uncharged residues, made at a single residue (Gly 166); cation-exchange retention data have been reported previously.22 This serves as a suitable case for exploring contributions of individual residues to protein-adsorbent affinity, whereas the cytochrome c variants examined previously show the combined effects of multiple substitutions. A somewhat higher degree of variation is examined in a pair of related heparin-binding proteins, the fibroblast growth factors FGF-1 and FGF-2, which have substantial sequence homology and structural similarities but differ appreciably in net charge at pH 7. The retention of these two proteins on a set of cation exchangers exhibits pronounced sensitivity to the stationary phase, which can be categorized into two different limiting results: strong retention of both FGF-1 and FGF-2 but low selectivity between these two proteins or comparatively lower overall retention with much greater binding differences between the FGFs.23 The varying trends in the retention of FGFs on different adsorbents indicate that distinct binding mechanisms, partly tuned by the physicochemical properties of the adsorbents, may contribute to the changes in FGF retention behavior on different adsorbents.23 Therefore, the retention of FGF-1 and FGF-2, which differ fairly extensively in structure, can be used in the investigation of how protein structure, combined with adsorbent properties, affects the binding mechanism and thus the retention extent. In the limit of the effect of substantial structural differences is the retention of two completely different proteins, lysozyme and cytochrome c. These proteins are similar in size and have approximately the same net charge at pH 7, but lysozyme was consistently retained much more strongly than cytochrome c on a large set of cation exchangers.1 There have been numerous other observations of “abnormal” retention that cannot be explained by the protein net charge, including separation of proteins of the same net charge and retention of proteins on like-charged adsorbents.3,18,22,24,25 However, the underlying retention mechanisms, and the basis for the retention differences among such independent proteins, remain incompletely understood, so the comparison presented here of lysozyme and cytochrome c represents a case study for such systems. THEORY AND COMPUTATIONAL METHODS The theory and methods used were described in detail in the companion paper18 and are therefore discussed only briefly here, particularly to elucidate those aspects peculiar to the proteins used in this study or to the quantities calculated for some cases. Protein Structures. Protein structures were obtained from the Protein Data Bank (PDB)26 for subtilisin wild type (2st1.pdb), (22) Chicz, R. M.; Regnier, F. E. Anal. Chem. 1989, 61, 2059-2066. (23) DePhillips, P.; Lenhoff, A. M. J. Chromatogr., A 2004, 1036, 51-60. (24) Rounds, M. A.; Regnier, F. E. J. Chromatogr. 1984, 283, 37-45. (25) Lesins, V.; Ruckenstein, E. Colloid Polym. Sci. 1988, 266, 1187-1190. (26) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. F.; Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112, 535-542.
2158
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
chicken egg white lysozyme (1lyz.pdb), horse heart cytochrome c (1hrc.pdb), FGF-1 (1jqz.pdb), and FGF-2 (2bfh.pdb). The 3D structures of the subtilisin mutants were constructed using MODELLER27 with the wild type subtilisin structure as the template. As the N-terminal residues of the FGFs have been found not to interact specifically with the remainder of the molecules,28 residues 1-18 of FGF-2, which were not resolved from the density maps, were neglected in modeling electrostatic interactions, under the assumption that they do not contribute much to differential retention features. Electrostatic Modeling. The approach used to calculate electrostatic interaction energies is a continuum one with the electrostatic potential assumed to be governed by the Poisson and Poisson-Boltzmann equations.29-32 Details of the equations and computational methods were described in the previous paper.18 These computations, performed at conditions corresponding to an ionic strength of 0.1 M, yield the electrical potential as a function of position, from which the interaction free energy (IFE) can be calculated for the protein and a charged end group, which represents the adsorbent. Due to the heterogeneous charge distribution and the anisotropic shape of the protein, the IFE is configuration-dependent, and an extensive sampling of the configurational space is necessary to correlate accurately the individual IFEs with the overall retention behavior. This is done via integration over the full configuration space to obtain the capacity factor k′ as18
A k′ ) FsVparticle A0
∫∫ Ω
∞
r0
(e-∆G1j(r,Ω)/RT - 1)r2 dr dΩ 2V0
(1)
where Fs is the number of end groups per unit particle volume, Vparticle the total particle volume in a packed column, V0 the retention volume of an unbound solute, and A/A0 the fraction of pore area that is accessible to a protein-sized solute. The angular space Ω represents orientations that are accessed in protein adsorption, which is assumed to include all sterically permitted configurations. However, the preference for adsorption in any given orientation is explicitly accounted for by the Boltzmann weighting e-∆G/RT. Because of the computational expense of the full configurational exploration required to evaluate eq 1, more economical but informative measures of relative retention are desirable. Since electrostatic interactionsspredominantly attractive in the systems of interestsbecome weaker with increasing separation distance r, the orientational integral at a short separation distance, r0,
IΩ(r)r0) )
∫ [e
-∆G(Ω,r)/RT
- 1] dΩ
(2)
can serve as a representative quantity characterizing retention differences among proteins.18 In general, Ω is integrated over all (27) Sali, A. Mol. Med. Today 1995, 1, 270-277. (28) Zhu, X.; Komiya, H.; Chirino, A.; Faham, S.; Fox, G. M.; Arakawa, T.; Hsu, B. T.; Rees, D. C. Science 1991, 251, 90-93. (29) Gilson, M. K.; Sharp, K. A.; Honig, B. H. J. Comput. Chem. 1988, 9, 327335. (30) Harvey, S. C. Proteins 1989, 5, 78-92. (31) Davis, M. E.; McCammon, J. A. Chem. Rev. 1990, 90, 509-521. (32) Sharp, K. A. Curr. Opin. Struct. Biol. 1994, 4, 234-239.
Table 1. Comparison of Orientational Integral, ∫Ωlocal[e-∆G(Ω,z)/KT - 1] dΩ, with the Retention Results of Subtilisin Variantsa variant
retention time (min)
orientational integral
G166D G166E wild type G166R G166K G166P G166V G166Y
7.10 7.55 8.30 11.90 12.05 18.15 18.30 18.40
-0.57 -0.54 -0.30 0.42 1.33
a The orientational integral was calculated for atoms within a radius of 15 Å of the R-carbon atom of the residue at position 166.
sterically accessible values, but the integral in eq 2 may also be evaluated over a more restricted domain in order to explore the local effect due to a particular region of the protein surface, e.g., one involved in specific binding. For these calculations, the separation distance between the protein and the end group, r0, was chosen to have a value of 2 Å between the surfaces of the protein and the end group. The IFE values at such a short separation distance are expected to provide a representative measure of the protein-adsorbent interaction in each orientation.18 For a comparative evaluation of protein binding, we use this orientational integral to compare the relative retention extents of different proteins. PROPERTIES OF PROTEINS AND ADSORBENTS Proteins. (1) Subtilisin. Subtilisin is a protease that has a broad specificity for proteins and is thus used as a constituent of laundry detergents.33 A large number of subtilisin variants have been engineered to achieve given protein specificities.34-39 Subtilisin mutants with single substitutions by charged or uncharged residues at residue 166, including G166K, G166R, G166E, G166D, G166P, G166V, and G166Y, were employed by Chicz and Regnier22 to study the contribution of individual amino acids to protein ionexchange chromatography. The retention of mutants was studied by elution in a 30-min linear gradient from 0 to 0.15 M NaCl, at pH 5, on a Mono-S cation-exchange column (Table 1).22 Residue 166 is in the substrate binding cleft accessible to solvent,35 and this residue was adjudged to make direct electrostatic contact with the ligands in cation-exchange chromatography.22 Secondary modes of interaction that cannot be accounted for by purely electrostatic calculations presumably contribute to the much higher binding extents for some variants, e.g., the hydrophobic/ uncharged polar substituted ones (G166P, G166V, G166Y). (33) Durham, D. R. J. Appl. Bacteriol. 1987, 63, 381-386. (34) Robertus, J. D.; Alden, R. A.; Kraut, J. Biochem. Biophys. Res. Commun. 1971, 42, 334-339. (35) Estell, D. A.; Graycar, T. P.; Miller, J. V.; Powers, D. B.; Burnier, J. P.; Ng, P. G.; Wells, J. A. Science 1986, 233, 659-663. (36) Wells, J. A.; Powers, D. B.; Bott, R. R.; Graycar, T. P.; Estell, D. A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1219-1223. (37) Wells, J. A.; Powers, D. B.; Bott, R. R.; Katz, B. A.; Ultsch, M. M.; Kossiakoff, A. A.; Power, S. D.; Adams, R. M.; Heyneker, H. H.; Cunningham, B. C.; Miller, J. V.; Graycar, T. P.; Estell, D. A. In Protein Engineering; Oxender, D. L., Fox, C. F., Eds.; Alan R. Liss: New York, 1987; pp 279-287. (38) Takagi, H.; Ohtsu, I.; Nakamori, S. Protein Eng. 1997, 10, 985-989. (39) Bryan, P. N. Biochim. Biophys. Acta 2000, 1543, 203-222.
However, pronounced local effects involving electrostatic contributions are apparent in the chromatographic discrimination for substitutions that conserve charge, viz. G166K versus G166R and G166D versus G166E. The importance of local structural details for overall retention is impressively seen in that the G166D and G166E variants, differing in only one methylene group, can still be differentiated chromatographically. (2) FGF-1 and FGF-2. FGF-1 and FGF-2 belong to the fibroblast growth factor (FGF) family that plays an active role in the modulation of cell proliferation and differentiation.28 This family exhibits high affinity toward heparin,40 and a wide range of investigations have been carried out on the heparin binding domains. FGF-1 and FGF-2 share ∼55% sequence identity (Figure 1).41 In FGF-1 N18 and a concentrated region of positive charges, composed largely of K112-K118, have been found to be the potential heparin binding site.42,43 In FGF-2, N27, K119, R120, K125, K129, and K135 were considered the leading potential contributors to the heparin binding,44-46 while further molecular modeling identified K26, R81, T121, Q123, and Q134 as being implicated as well.47 The alignment of the two FGFs (Figure 1) shows that the proposed binding residues have counterparts in each protein, suggesting that FGF-1 and FGF-2 may bind to heparin in a similar manner. Despite their similarities, the two proteins have significant differences in properties that play important roles in determining electrostatic interactions, such as isoelectric point and the number and distribution of charged amino acid residues (Table 2). Isocratic retention of FGF-1 and FGF-2 at pH 7 in 10 mM phosphate buffer with different NaCl concentrations was measured on a set of cation exchangers.23 Both FGFs displayed strong retention on SP Spherodex, Cellufine Sulfate, and EMD SO3- M, and much weaker retention on SP-550 C, SP Sepharose FF, and SP-650 M. On the materials with strong retention, the two proteins had very similar capacity factor values. For the Tosoh and Sepharose materials that exhibit lower retention of FGFs, however, the differences between the two proteins are much more significant, ranging from 3- to 17-fold higher retention of FGF-2 than of FGF-1. (3) Cytochrome c and Lysozyme. Whereas the proteins discussed above are highly homologous, the final case that we examine is the retention of two completely different proteins, horse cytochrome c and chicken egg lysozyme, in cation-exchange chromatography. The charge and size properties of these two proteins are listed in Table 3. Both are similar in size and carry similar charges at pH 7, although significant differences in secondary structure exist between these two unrelated proteins. Isocratic retention of both proteins was measured at pH 7 in 10 (40) Gospodarowicz, D.; Cheng, J. J. Cell. Physiol. 1986, 128, 475-484. (41) Ogura, K.; Nagata, K.; Hatanaka, H.; Habuchi, H.; Kimata, K.; Tate, S.; Ravera, M. W.; Jaye, M.; Schlessinger, J.; Inagaki, F. J. Biomol. NMR 1999, 13, 11-24. (42) Blaber, M.; DiSalvo, J.; Thomas, K. A. Biochemistry 1996, 35, 2086-2094. (43) Pineda-Lucena, A.; Jimenez, M. A.; Nieto, J. L.; Santoro, J.; Rico, M.; GimenezGallego, G. J. Mol. Biol. 1994, 242, 81-98. (44) Eriksson, A. E.; Cousens, L. S.; Weaver, L. H.; Matthews, B. W. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 3441-3445. (45) Zhang, J. D.; Cousens, L. S.; Barr, P. J.; Sprang, S. R. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 3446-3450. (46) Ago, H.; Kitagawa, Y.; Fujishima, A.; Matsuura, Y.; Katsube, Y. J. Biochem. (Tokyo) 1991, 110, 360-363. (47) Thompson, L. D.; Pantoliano, M. W.; Springer, B. A. Biochemistry 1994, 33, 3831-3840.
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
2159
Figure 1. Sequence alignment of FGF-1 and FGF-2. Boldface type residues are the proposed heparin binding sites.42,44,47
Table 2. Properties of FGF-1 and FGF-2
molecular mass (kDa) calculated pIa amino acid composition Arg Lys His Asp Glu calculated net charge at pH 7
FGF-1
FGF-2
15.8 7.9
16.4 9.6
6 11 5 7 9 1.5
11 14 3 7 8 10.2
a Calculated pI values from the Expert Protein Analysis System server of the Swiss Institute for Bioinformatics.
Table 3. Properties of Chicken Egg White Lysozyme and Horse Heart Cytochrome c
equivalent radius (Å)a calculated pIb calculated net charge at pH 7 number of arginine residues number of lysine residues
lysozyme
cytochrome c
15.9 9.3 8.1 11 6
15.2 9.5 8.3 2 19
a
Radius of sphere with equivalent volume of the protein. b Calculated pI values from the Expert Protein Analysis System Server of the Swiss Institute for Bioinformatics (SIB).
mM phosphate buffer with different NaCl concentrations.1 Despite the similarities in size and charge, lysozyme displays significantly higher retention than cytochrome c on a wide range of cation exchangers.1 This further shows that simple net charge and size arguments are not adequate for the characterization of protein retention and that further detailed structural information must be included to model the process. Stationary Phases. Although cytochrome c variants18 and lysozyme versus cytochrome c1 show consistent retention trends on the adsorbents studied, FGF-1 and FGF-2 display significant differences in retention and selectivity across a set of cation exchangers.23 Consequently, although the main purpose of this study is to investigate the role of protein structure in adsorption, the structural and chemical properties of the adsorbent are essential for evaluating protein-adsorbent interactions. The morphological, chemical, and mechanical properties of the materials used here differ appreciably, and these detailed physicochemical properties are potentially important in determining retention behavior. 2160
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
Toyopearl SP-550 C and SP-650 M (Tosoh Biosep, Montgomeryville, PA) are based on a methacrylate matrix derived from Toyopearl HW materials and functionalized with sulfonated propyl groups. SP Sepharose FF (GE Healthcare, Piscataway, NJ) is a 6% highly cross-linked agarose material with ion-exchange groups of 3-(2-hydroxypropoxy)-1-propanesulfonic acid. EMD Sulfate obtained from EMD Chemicals Inc. (Gibbstown, NJ) is a strong cation-exchange material also based on the Toyopearl HW base matrix and functionalized with “tentacle” functionalities. The functional ion-exchange groups are bonded via polyelectrolyte chains that are thought to allow the ionic groups to adopt a configuration optimal for protein-adsorbent electrostatic interaction and high capacity. SP Spherodex from Ciphergen (Fremont, CA), a strong cation-exchanger, has a silica-dextran composite base matrix with methyl sulfonate as the functional group. Cellufine Sulfate from Amicon (Beverly, MA) is based on rigid cellulose with a 3000 molecular weight exclusion limit. It is derivatized to have a low concentration of sulfate ester functional groups.
RESULTS AND DISCUSSION In the following, our results of modeling protein retention are compared with the experimental observations to provide a basis for elucidating protein binding mechanisms. The orientational integral, IΩ, is evaluated from eq 2 in the configuration space defined as all sterically permitted angular positions at a separation distance of 2 Å between the protein surface of interest and the surface of the functional group. In some cases, the full orientational space is included, but in others, the integral is over a more localized region. Subtilisin Variants. The dominance of short-range electrostatics in determining protein retention, as seen for cytochrome c variants,18 can be examined even more critically for single-site mutants. Only 1 residue out of 275 varies in the subtilisin mutants, and comparison of the crystal structures of these residue 166 mutants indicates that structural changes induced by the substitutions are localized rather than global.48 Therefore, it is sufficient to investigate just the local configurational space near the substituted residue. The region within 15 Å of the R-carbon of the residue at position 166 was included in the sampling of local (48) Bott, R.; Ultsch, M.; Wells, J.; Powers, D.; Burdick, D.; Struble, M.; Burnier, J.; Estell, D.; Miller, J.; Graycar, T.; Adams, R.; Power, S. In Biotechnology in Agricultural Chemistry; LeBaron, H. M., Mumma, R. O., Honeycutt, R. C., Duesing, J. H., Phillips, J. F., Haas, M. J., Eds.; ACS Symposium Series 334; American Chemical Society: Washington, DC, 1987; pp 139-147.
angular orientation, Ωlocal, defined by the Connolly critical points49,50 in evaluating the orientational integral. The mutants studied can be divided into several groups. G166D and G166E were successfully separated by gradient elution on a Mono-S column,22 a remarkable result considering the small structural difference versus the >6% change in retention time in gradient elution (Table 1). The interaction energies for the end group with these two variants in the region around residue 166 are mostly repulsive because of the negative charges on both the residue and the end group. The values of the local orientational integral for these two proteins are -0.57 and -0.54, respectively (Table 1), showing that substituting Glu by Asp produces slightly stronger repulsion between the protein and the end group around residue 166. Residue 166 is located at the bottom of a large open binding cleft, which might allow projection of a longer side chain at this position into the cleft and thus closer to the surface.35 As a result of the accessibility of the end group to the cleft, G166E is predicted to experience stronger repulsion in a number of orientations near residue 166. However, a more significant effect may be that of Lys170, the only basic residue within 10 Å of residue 166; the atoms around the -amino group in G166E are calculated to have IFEs on the order of a few tenths of a kT less than the corresponding ones in G166D, the key reason being that the negative charge at the end of Glu166 extends further away from Lys170. Analysis of electron density maps of G166N and G166K indicates that there is some conformational variability at position 166, and such structural disorder can also affect binding strength on ion exchangers.48 It is possible that the local change in geometry due to the additional methylene group also produces a difference in nonelectrostatic contributions to the interaction. The positively charged amino acids, Arg and Lys, on the respective substituted variants, G166R and G166K, are oppositely charged to the adsorbent, and both display attractive interactions toward the end group at this orientation. The calculated orientational integral is, however, much higher for G166K, suggesting that this mutant should be much more strongly retained. However, G166K has only a slightly longer retention time than G166R (∼1%) (Table 1).22 This apparent discrepancy for the G166R and G166K variants is attributable to the difference between Arg and Lys. A study of heparin binding to oligopeptides of arginine and lysine showed that binding to arginine peptides was significantly stronger than that to lysine peptides.51 Such stronger binding of Arg than Lys would not be expected based on continuum electrostatics, as in our calculations. Whatever the origin of the stronger binding of Arg than Lys, it would be expected to reduce the significantly higher retention predicted for G166K than for G166R by the electrostatic model and, at least qualitatively, explain the measured smaller retention difference between G166R and G166K. The arginine-lysine binding difference can be rationalized in terms of hard and soft acid and base concepts,52 and the mechanistic basis can be attributed to hydration effects.53,54 The (49) Connolly, M. L. J. Appl. Crystallogr. 1983, 16, 548-558. (50) Lin, S. L.; Nussinov, R.; Fischer, D.; Wolfson, H. J. Proteins 1994, 18, 94101. (51) Fromm, J. R.; Hileman, R. E.; Caldwell, E. E. O.; Weiler, J. M.; Linhardt, R. J. Arch. Biochem. Biophys. 1995, 323, 279-287. (52) Pearson, R. G. J. Chem. Educ. 1968, 45, 643-648. (53) Collins, K. D. Biophys. J. 1997, 72, 65-76. (54) Asthagiri, D.; Schure, M. R.; Lenhoff, A. M. J. Phys. Chem. B 2000, 104, 8753-8761.
hydration energy of a polar entity in solution depends on the charge density, with kosmotropes, ions with high charge density, having a strong water binding capacity.53 It has been shown that the local hydration structure of a peptide surface depends on the properties of the amino acid residue,55,56 with more ordered water molecules around polar residues. The release of structured water around the protein and ligand surfaces upon adsorption results in an increase in the entropy of the system, and these effects are difficult to quantify.57,58 For instance, detailed calculations suggest that a solvent-separated minimum can exist in the potential of mean force between two cavities,59 also indicating the importance of explicit description of water molecules. The heterogeneous structure of the hydration layer and its changes contribute significantly to the adsorption energy. However, the electrostatic approach used here, in which the solvent is described as a continuum, cannot account for these contributions due to the detailed structure of the hydration layer. The nonpolar replacement variants G166V and G166P and the uncharged polar substituted G166Y were found to have substantially longer retention times than that of the wild type (Table 1), which, not surprisingly, cannot be explained by electrostatic energy considerations. Both hydrophobic binding and structural disruptions due to the bulkiness of Tyr and the conformational limitations of Pro relative to the native Gly may be contributing factors. FGF-1 and FGF-2. The IFEs for the two FGFs in the orientations that are most favorable energetically (IFE < -2.6kT at a 2-Å separation distance) are listed in Table 4. For both FGF-1 and FGF-2, the heparin binding sites exhibit relatively strong negative IFEs. Compared to the IFE landscape of the cytochrome c variants,18 the free energy distributions for the FGFs include several orientations demonstrating much more favorable interaction energies at a comparable separation distance (several approaching -6kT vs almost none below -4kT). As electrostatic interactions decrease sharply with the separation distance between the interacting charges, the charge distance map, indicating the distance between the charge on the end group and those on the protein, provides a basis for interpreting the configurationaldependent electrostatic interactions. It was shown in our earlier modeling of cytochrome c that one positive charge very close to the adsorbent end group can enhance the protein attraction to the end group significantly and that this effect is amplified near multiple positive charges.18 The FGFs have more locally concentrated distributions of positive charges than do the cytochrome c’s, specifically around the heparin binding site, and most of the negative charges are relatively far from the favorable basic residues (Figure 2). This is the likely explanation for the extremely negative values of the interaction energy (Table 4) and may result in strong enhancement of FGF binding via the highly positively charged regions. The energetically favorable orientations with IFE values below the threshold value of -2.6kT are mostly located in positive patches, the majority of which are situated within or near the (55) Komeiji, Y.; Uebayasi, M.; Someya, J.; Yamato, I. Proteins 1993, 16, 268277. (56) Song, D.; Forciniti, D. J. Chem. Phys. 2001, 115, 8089-8100. (57) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1979, 71, 350-366. (58) Norde, W. Macromol. Symp. 1996, 103, 5-18. (59) Hummer, G.; Garde, S.; Garcia, A. E.; Paulaitis, M. E.; Pratt, L. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1552-1555.
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
2161
Table 4. Most Favorable Configurations (IFE < -2.6kT) Calculated for FGF-End Group Interactionsa FGF-1
FGF-2
atom
IFE (kT)
atom
IFE (kT)
atom
IFE (kT)
N18 CG K112 CD K112 NZ K113 CB K113 NZ N114 N N114 CG K118 CA K118 CG K118 CE K118 NZ R122 O R122 CB R122 CD Q127 CA Q127 CG Q127 NE2 K128 N K128 CB A129 N V137 N
-5.41 -3.44 -3.32 -2.84 -3.89 -3.40 -4.54 -3.63 -3.25 -4.35 -3.79 -3.02 -4.69 -2.71 -3.09 -2.79 -4.05 -4.01 -4.78 -5.81 -2.67
R22 NH1 R22 NH2 Y24 CD2 K26 O K26 CG N27 CG N27 ND2 G28 N G38 C K46 CE A84 O S85 CA K86 N K119 CA K119 CB K119 CG K119 CD K119 CE K119 NZ R120 O R120 CB R120 CG R120 NE
-3.21 -2.88 -2.99 -2.74 -2.64 -5.35 -3.70 -3.92 -2.69 -2.83 -2.86 -2.93 -2.69 -5.96 -5.24 -5.82 -5.17 -3.06 -2.88 -2.60 -2.84 -3.93 -5.65
R120 CZ T121 C T121 CG2 Q123 CB Y124 O Y124 CD2 Y124 CE1 Y124 CZ K125 CD K125 CE K125 NZ K129 CE K129 NZ Q134 CA Q134 CB Q134 CD K135 N K135 CB K135 NZ A136 N A136 CB K145 N K145 CA S146 O
-2.79 -2.63 -3.23 -2.96 -3.02 -3.50 -3.33 -2.70 -5.65 -4.32 -3.84 -2.94 -2.90 -3.28 -3.90 -3.68 -3.79 -4.19 -3.35 -5.79 -6.05 -3.51 -2.78 -2.66
a The orientations refer to the position of the end group, 2 Å from the protein surface at a location defined by a particular atom on the surface; e.g., N18 CG refers to the atom CG in residue N18. Residues in boldface type are part of the heparin binding site.42,44,47
Figure 2. Charge distance maps of orientations that have the most favorable interaction free energies, viz. N18 CG in FGF-1 (hatched, IFE ) -5.3kT), N27 CG in FGF-2 (0, IFE ) -5.3kT), and G84 O in cytochrome c (9, IFE ) -4.1kT).
heparin binding sites (Figure 3). This reflects the high density of positive charges in the region of the heparin binding sites on the FGFs.60 Equivalent residues within the heparin binding sites in 2162 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
the two FGFs exhibit similar interaction free energies (Table 4 and Figure 1), demonstrating the functional similarity of the binding site components in FGF-1 and FGF-2. However, a number of binding site residues in FGF-2, such as K119, R120, K125, and A136, display noticeably higher affinity than the corresponding binding site residues in FGF-1. A close look at the charge microenvironment around the related residues on the two proteins shows that in FGF-2 the sites are located in a more concentrated basic region than their counterparts in FGF-1, such as atom K112 CD in FGF-1 versus K119 CA in FGF-2 shown in Figure 4. For FGF-2, the favorable configurations also include a number of basic and uncharged residues that do not fall within the main heparin binding pocket, such as R22, Y24, K46, A84, S85, and K145. These residues are concentrated as two clusters on the molecule (Figure 3) and might represent additional binding regions participating in FGF adsorption. Two possible limiting cases were proposed for FGF binding to different stationary phases to explain the different patterns of retention.23 The first scenario is nonspecific adsorption that explores all feasible configurations. There are appreciable net charge differences between FGF-1 and FGF-2 (+1.5 vs +10 at pH 7) that directly affect the charge locations and overall net charge (Table 2, Figure 1). Thus, if the end group has the freedom to explore most of the charges on the protein, one would expect a retention difference due to the substantial differences in the charged residue compositions, with most positive charges participating in the interactions either directly or by helping to provide a more favorable charged microenvironment. Consequently, FGF-2 should exhibit a much higher affinity than FGF-1, which corresponds to the clear selectivity between FGF-1 and FGF-2 observed on Tosoh and Sepharose materials. The other limiting situation describes a more selective process, in which FGF is bound in a small number of configurations, perhaps due to the structural and charge complementarities of the protein-adsorbent system. It has been observed that proteins are more strongly retained on adsorbents with a significant fraction of the pore space being of size comparable to that of the protein,1 possibly because this allows more extended contact between the protein and the surface, hence promoting electrostatic interaction. In addition, heparin binding was found to require, other than a specific pentasaccharide sequence in the heparin,61 a characteristic spacing and basic charge density in the heparin binding protein.60 Such charge and structural complementarity can be extended to the protein-adsorbent system here, which has the potential for a cooperativity effect, viz. multiple charge-charge interactions between the protein and the adsorbent. This can amplify locally extreme attractive interactions at certain orientations, which would then become the major binding conformations that determine the resulting extents of retention. Based on these two binding mechanisms, we calculated the orientational integrals over the corresponding configurational spaces. The contribution averaged over the whole configurational space of FGF-2 is twice that of FGF-1 (Table 5), in reasonable agreement with the experimental k′ differences on adsorbents displaying selectivity.23 Because of the Boltzmann weighting in (60) Margalit, H.; Fischer, N.; Bensasson, S. A. J. Biol. Chem. 1993, 268, 1922819231. (61) Guimond, S.; Maccarana, M.; Olwin, B. B.; Lindahl, U.; Rapraeger, A. C. J. Biol. Chem. 1993, 268, 23906-23914.
Figure 3. Calculated favorable residues (shown in space-fill form) in FGF-1 and FGF-2 including most of the heparin binding site residues identified from previous studies of FGF binding,42,44,47 shown in blue. The residues in orange are calculated to have strong negative interaction energies but are not located in the heparin binding domain.
Figure 4. Charge environments of K112 CD in FGF-1 and K119 CA in FGF-2 at the orientations defined by atoms shown in orange, corresponding to IFE values of -3.44kT and -5.96kT in FGF-1 and FGF-2, respectively. Positive charges that are within 10 Å of this configuration are in blue. K112 CD in FGF-1 has only three positive charges, the NZ atoms in K112, K113, and K118 within 10 Å, while K119 CA in FGF-2 is located in a region with a higher local concentration of positive charges, with five basic residues, R44, R120, K125, K129, and K135, within 10 Å.
Table 5. Orientational Integral, IΩ, of FGF-1 and FGF-2 at a 2-Å Separation Distance, Averaged over Different Orientational Spaces
full moleculesa heparin binding sitesb favorable binding configurationsc
FGF-1
FGF-2
3.6 63 63
7.6 93 81
a All sterically permitted orientations were sampled. b Integrated over the heparin binding sites. c Averaged over the favorable orientations with IFE < -2.6kT.
eq 2 coupled with the highly negative IFE values around the heparin binding site (Table 4), the calculation over the full orientational domain is strongly influenced by the binding site IFEs. However, the integrals for configurations confined only to
the heparin binding sites or the favorable binding residues as determined by the IFE calculations were also calculated and show the FGF-2 value to be ∼50% higher than that of FGF-1 for the heparin binding site case versus 30% higher for the favorable residue case, a larger difference than seen experimentally on the unselective adsorbents.23 There are several possible factors in the adsorbent physicochemical properties that may give rise to the retention patterns seen experimentally,23 given the calculated electrostatic IFEs. The Toyopearl materials SP-650 and SP-550 have comparatively rigid methacrylate base matrices with propyl spacer arms. The agarose base matrix of SP Sepharose FF is macroscopically somewhat compressible, but because it is made up of bundles of agarose fibers,62 it may not be very flexible locally. This factor, and the seven-atom spacer arm used, can hinder the possibility of binding Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
2163
over an extended part of the protein surface. These characteristics make high-affinity binding less probable. On the other hand, SP Spherodex, Cellufine Sulfate, and EMD SO3- consist of sulfated end groups attached to more flexible base matrices or to polymer chains, which may resemble the heparin polysaccharide structure to some extent and have the potential of inducing binding over an extended region analogous to that seen for heparin. While the orientational integrals over different domains qualitatively capture the two limiting types of retention and selectivity behavior, the modeling results do not account adequately for the high-affinity binding mechanism, considering the very similar experimental retention times for strongly bound FGFs as opposed to the ∼30% difference found for calculations, assuming the selective adsorption pattern. The postulated multipoint interactions could provide a plausible resolution, especially given the very strong overall retention observed, but because they cannot be defined more explicitly with any confidence, they were not considered in our calculations. The modeling did not consider residues 1-18 in FGF-2, which includes three charged residues, E5, D6, and K18. Of these, K18 would be expected to strengthen the predicted retention of FGF2, which would be consistent with the observed results on the selective stationary phases. However, this residue is also closest to the heparin binding site and thus may play a significant role in the heparin-analogous binding, resulting in even stronger predicted retention of FGF-2, aggravating the already overestimated retention of FGF-2 relative to that of FGF-1. Thus, the omission of residues 1-18 is unlikely to be the principal origin of the modeling versus experiment discrepancies seen for the FGFs. Although most of the binding site residues are positively charged, and the importance of electrostatic contributions to FGF binding even to heparin is evidenced by easier elution of heparinprotein complexes at high salt,63 energetic characterization of mutated FGFs47 showed that only ∼33% of the overall retention resulted from electrostatic contributions, with uncharged residues such as N27 and Q134 responsible for >33% of the binding free energy. This suggests that other intermolecular forces may comprise most of the remaining binding free energy. One contribution to this may be the entropic one due to displacement of water from the heparin binding cleft, which may occur over an extended length of the cleft even though the electrostatic interactions holding heparin in the cleft are relevant only over a much shorter length. Lysozyme and Cytochrome c. The orientational integrals over all accessible orientations at 2-Å separation distance predict the retention of lysozyme on the negatively charged surface to be much lower than that of cytochrome c (1.0 for lysozyme vs 3.4 for cytochrome c). This is diametrically opposed to experimental observations, where lysozyme consistently has much higher retention factors at all salt concentrations and on all stationary phases studied.1 As the structural details of the protein, both geometric and electrostatic, have been incorporated in the electrostatic model, the unsuccessful prediction at this level of modeling raises the question of which other molecular interactions must be included (62) Arnott, S.; Fulmer, A.; Scott, W. E.; Dea, I. C. M.; Moorhouse, R.; Rees, D. A. J. Mol. Biol. 1974, 90, 269-284. (63) Seno, M.; Sasada, R.; Kurokawa, T.; Igarashi, K. Eur. J. Biochem. 1990, 188, 239-245.
2164
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
to characterize protein retention successfully. A comparison between the amino acid compositions of lysozyme and cytochrome c reveals a significant difference in the makeup of the basic residues that provide the positive charge, i.e., the number of arginine and lysine groups in the molecules (Table 3): lysozyme has 11 arginines while cytochrome c has only 2, although the net charge at pH 7 is approximately the same for both.1 In light of the same argument as that for the retention discrepancy in the subtilisin G166K/G166R variants, stronger binding of arginine than lysine to anions, which is not accounted for in the model, is one reason for the disagreement between experiment and modeling. Combined underestimation of the binding of a large number of arginine residues in lysozyme may result in the calculated retention order of lysozyme/cytochrome c being opposite to that seen experimentally. These results underline the well-recognized role possible for other interaction mechanisms in modulating significantly the electrostatic factors that dominate protein retention in ionexchange chromatography. Properly accounting for these effects is essential for satisfactory molecular modeling of protein adsorption in IEC, but probing them directly is problematic. For two distinct contributions ∆G1 and ∆G2 to the overall IFE ∆G, the Boltzmann-weighted contribution to retention e-∆G/RT ∼ e-∆G1/RTe-∆G2/RT. Therefore. for strong electrostatic interactions given by ∆G1, a much weaker effect ∆G2 can still introduce significant amplification or attenuation that has a measurable impact on retention, even if this secondary interaction is not strong enough to be noticeable in the absence of the strong electrostatic interaction. Hydration effects appear to be especially important for improved retention modeling, as these effects appear to be manifested in multiple systems and continuum descriptions of the solvent do not capture the dynamics of the hydration structure. Molecular dynamics simulations, with detailed information on the protein and explicit description of water molecules,64 are needed to address the protein-solvent interaction and its contribution to protein adsorption more completely. CONCLUSIONS We have shown that electrostatic modeling with molecular details can account to varying degrees of accuracy for the small differences in retention trends within protein groups. The systems studied, with increasing numbers of mutations ranging from single-substituted subtilisin variants to cytochrome c variants (e6 substitutions)18 to FGFs with significantly greater structural differences, show that electrostatic interactions adequately capture the principal effect in several situations, especially when the number of mutations is limited. Additional effects such as hydration can fine-tune this dominant contribution, but a reasonable picture of at least the elution order can emerge. For proteins with more significant structural variations, as in the lysozymecytochrome c comparison, the accumulation of uncertainties due to other molecular forces can make even qualitative predictions of trends unreliable. An additional factor that emerges from the results is the synergistic role of the protein structure and the stationary-phase structure and properties. The subtle role of this effect is seen in the FGF results, which suggest that multipoint attachment may (64) Bujnowski, A. M.; Pitt, W. G. J. Colloid Interface Sci. 1998, 203, 47-58.
be prevalent on some adsorbents. Because this depends on the distribution of charges on the protein and the distribution of ligand spacing on the adsorbent, the effect is also very difficult to account for in any modeling effort even if the underlying interactions are predominantly electrostatic and thus nominally amenable to calculation using continuum methods. A possible initial approximation would be to augment the IFE calculated for each configuration as in the current work with a mean-field contribution due to additional points of interaction. However, such effects are likely to be confounded with those discussed in the previous paragraph and may therefore be difficult to resolve.
ACKNOWLEDGMENT We are grateful to Dr. Donald Bashford for making the MEAD package available and to Dr. Dilipkumar Asthagiri for helpful discussions. This work was supported by the National Science Foundation (Grants CTS-9977120 and CTS-0350631). Received for review August 25, 2004. Accepted January 13, 2005. AC048733F
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
2165
