The dynamics and binding of a Type III antifreeze protein in water and on ice

THEO CHEM ELSEVIER

Journal of Molecular Structure (Theochem) 388 (1996) 65-77

The dynamics and binding of a Type III antifreeze protein in water and on ice Jeffry D. Madura a'*, Mark S. Taylor a, Andrzej Wierzbicki a, John P. Harrington a, C.S. Sikes b, Frank S6nnichsen c aDepartment of Chemistry and bDepartment of Biological Sciences, University of South Alabama, Mobile, AL 36688, USA CProteinEngineering Network of Centres of Excellence Department of Biochemistry, Universityof Alberta, Edmonton, Alta. T6G 2S2, Canada

Received 4 January 1996; accepted 25 March 1996

Abstract Certain plants, insects and fish living in cold environments prevent tissue damage from freezing by producing antifreeze proteins or antifreeze glycoproteins [1]. These proteins work by inhibiting ice growth below the normal equilibrium freezing point of water. Fish antifreeze proteins are categorized into helical Type I AFPs, cysteine rich globular Type II AFPs, and small globular Type III AFPs. A three dimensional structure of Type III antifreeze protein was determined recently by NMR spectroscopy [2]. Using this solution structure we performed a 110 ps molecular dynamics simulation in a 50 x 50 × 50 .~ box of TIP3P water. Structural, dynamical and protein-water interactions during the simulation were investigated and are reported in this article. In addition, the dynamics structure was used to investigate possible protein-ice binding interactions. In particular, the protein face proposed by Chao et al. [3] was modeled on the (100) ice face and is discussed in terms of protein-ice interactions. Keywords: Ice-binding protein; Molecular modeling; Hydrogen bonding; Protein-ice modeling; QUANTA; CHARMM

1. Introduction Many cold-water fish are able to avoid the freezing of their tissues by producing antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) [1]. These proteins prevent the freezing of body fluids in a noncolligative fashion; they depress the freezing point of their solutions without affecting the colligative melting point [4]. The resulting temperature gap or thermal hysteresis is often referred to as antifreeze activity and it is used to define the relative activities at * Corresponding author.

the same concentration of different antifreeze peptides. The freezing point depression is caused by direct binding of AFPs to the surface of ice crystal nuclei. The AFP binding apparently occurs predominately at the bipyramidal and prism ice faces [5] in specific orientations restricting ice growth normal to the binding surface, and it is this binding in conjunction with the Kelvin-effect which locally depresses the freezing point without altering the melting point [61. Fish AFPs are divided into several structurally distinct groups as shown in Fig. 1. The most extensively studied are the Type I AFPs which are generally

0166-1280/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S0166-1280(96)04622-2

66

Type I

J.D. Madura et al./Journalof Molecular Structure (Theochem) 388 (1996) 65-77

Type II

Type III

Fig. 1. MOLSCRIPT[21] ribbonrepresentationof the TypeI, TypeII and TypeIII fishantifreezeproteins.The Type I beinghelicalin nature, the TypeII being globularwith disulfidebridges (representedby yellowlines), and the Type III being globularwithoutdisulfidebridges. alanine-rich, a-helical peptides of 36-44 residues. It is accepted that regularly spaced polar and charged groups along the helix surface match similarly spaced oxygens of a specific orientation on particular ice planes [7]. This structural fitting allows for adsorption of the AFP via hydrogen bonding and consequential inhibition of the freezing process. Computer models of Type I AFPs have supported the general matchedspacing mechanism [7-9]. With the knowledge of the adsorption plane these studies not only identified sterically favorable interactions between the ice surface and the protein, but could also confirm the preferred orientation of Type I AFPs based on energetic interactions between the protein and the ice surface [7]. The helical antifreeze glycoproteins are thought to function in a similar manner in which disaccharides attached to the threonine side chains of their ALA ALA THR repeat units are able to bind with regular ice crystal surfaces [10]. Type II and Type III AFPs in contrast are non-helical molecules without repetitive

sequences, and because of their more irregular structures it is not clear whether the Type II and Type III bind in a similar manner as Type I AFP. Type II AFPs are large (120 + residues), cysteine-rich proteins composed of /~-sheets, reverse turns and helices. Currently, experimental three dimensional structural data are not available for Type II AFPs. However, a model for their tertiary fold was generated recently by comparative modeling on the basis of a sequence similarity between Type II AFPs and the carbohydrate recognition domain of C-type lectins [11]. Type III AFPs are of intermediate size, containing about 65 residues, and lack a predominant amino acid type. NMR studies have shown this protein (ocean pout, Macrozoarces americanus) to contain E-sheets, two anti-parallel triple E-strands and one anti-parallel double E-strand [2] (see Fig. 2). The structure also displayed a less refined extended loop region over 13 residues (residues 28-40). The protein surface was shown to be void of large hydrophilic areas, and

J.D. Madura et al./Journal of Molecular Structure (Theochem) 388 0996) 65-77

67

/

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Average

Fig. 2. MOLSCRIPT[21] ribbon representation of the Type llI protein, the NMR structure on the left and the average solution structure on the fight. The color scheme is as follows: the N-face is blue, the C-face is red, the loop region is green, and the two strand region is yellow. moreover, a regular spacing between hydrophilic residues was not apparent. Subsequently, site specific mutagenesis experiments have identified three residues important for the protein-ice interaction [3]. Also, to our knowledge, in contrast to most other AFPs, ice-etching studies on M a c r o z o a r c e s a m e r i c a n u s have not to date unambiguously established the exact adsorption plane for this Type III AFP, however, the most prominent ice face to which Type III AFPs bind is the primary prism face of ice (100) [51. In an attempt to elicit details of protein-water

interactions, we have conducted a molecular dynamics simulation on the Type III AFP in water and compare the molecular dynamics structure with the N M R structure (see Fig. 3). Additionally we have used our molecular modeling methods to test the proposed AFP binding surface of Chao et al. [3] to the (100) face of ice. For this study the N M R structure of a mutant (OAE) component derived from HPLC-12 of the ocean pout was solvated in a periodic box of water. The system was thermalized and equilibrated for a total of 55 ps and data collection was carried out for an additional 110 ps. Since the Type III protein

Fig. 3. Stereoview of the NMR (blue) protein backbone superimposed with the average solution (red) structure. The RMS difference for the backbone atoms shown between the NMR and average solution structure is 2.37/~.

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J.D. Madura et aL/Journal of Molecular Structure (Theochem) 388 (1996) 65-77

structure exhibits a wide distribution of hydrophobic and hydrophilic residues, computational methods were also used to explore the interaction of the Type III AFP with the (100) ice planes using the average simulation structure (see Fig. 4). The protein surface chosen to be studied in these interaction calculations is the proposed surface of Chao et al. [3].

2. Materials and methods 2.1. M o l e c u l a r dynamics

A NVE molecular dynamics simulation was performed on a system consisting of a single 66-residue Type III AFP; M NQASVVANQL IPINTALTLV MMRS EVVTPV GIPAEDIPRL VSMQVNRAVP LGTILMPDMV KGYAA solvated in a periodic box of TIP3P [12] water molecules. The protein used was the NMR structure of the QAE component (HPLC-12) from ocean pout. Polar hydrogens were added to the protein using QUANTA/ CHARMm 4.0 [13]. The protein was placed in a 50 x 50 x 50 A3 box of water and all water molecules with oxygens within 2.3 .A of the solute were removed. The solvated protein, consisting of 11991 atoms, was initially subjected to 100 steps of steepest descent energy minimization to relax the protein-water interactions by holding the protein fixed and allowing the waters to move freely. At the end of the minimization the RMS gradient was 2.883 and the total energy for the system was -41846.0kcal mol-1. The system, with no constraints, was then subjected to 5 ps of thermalization, increasing the temperature from 0 K to 300 K in 12 K increments, followed by 50 ps of equilibration. Finally, data was collected for 110 ps with coordinate, velocity and energy files being saved every 0.05 ps. A nonbonded cutoff of 12 A was used in the calculations and the non-bonded list was updated every 25 steps. The electrostatic and van der Waals potential energy terms were gradually switched to zero over the range of 9.0 to 11.0 A. All calculations were performed using CHARMM 22 [14] on an IBM RISC 6000 Model 350 and a Cray C90. Graphical analysis of the dynamics results and

molecular modeling were conducted on IBM RISC 6000 and Silicon Graphics workstations using QUANTA 4.0. 2.2. Protein~ice modeling

The protein/ice modeling was conducted using the averaged simulation protein structure on the (100) ice planes. The (100) ice plane was constructed from the fractional coordinates of ice using Cerius 2 [13]. The space group used for ice, Ih, was No. 194 P63/mmc, unit cell constants were a -- b = 4.516 ,A, c = 7.354/~, a = /~ = 90 °, 7 = 120°, and fractional coordinates used for ice were O(0.3333,0.6667,0.0629), Ha(0.3333,0.667,0.1989), and Hb(0.4551,0.9102, 0.0182). The binding energy, AEbindiag was calculated using the following expression A F"binding -~ g

complex - (E AFP + E ice)

where Ecomplexis the minimized energy of the complex, EAFP is the energy of the Type III AFP, and Ei,~ is the energy of the ice slab. The individual energy terms were calculated by minimizing all degrees of freedom except the ice oxygen atoms which were held fixed in their crystallographic positions. The minimization procedure consisted of 500 steps of steepest descent followed by 500 steps of ABNR. The protein was then re-docked by hand using the modeling tools in QUANTA 4.0 and the binding energy was recomputed. This process was repeated many times to locate the 'lowest' energy complex. Once the 'lowest' energy complex was located a systematic search for a lower energy complex was performed. This was done by taking the 'lowest' energy complex translating the AFP in the x and y directions by 0.5 A along the ice surface, running a short minimization of 200 ABNR steps followed by dynamics (2 ps) of the ice slab with the protein held fixed at 600 K, and then calculating the binding energy as described above. o

3. Results 3.1. A v e r a g e solution structure

Using the coordinate data sets collected during the dynamics simulation an average AFP solution

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.I.D. Madura et al./Journal of Molecular Structure (Theochem) 388 (1996) 65-77

structure was calculated. A stereo view of the averaged solution structure protein backbone superimposed upon the NMR protein backbone is given in Fig. 3. The stereochemical quality of the averaged structure was assessed using the analysis package PROCHECK [15] which indicates that stereochemically the structure can be favorably compared to a 2 A resolution X-ray structure (Fig. 5(a)). As expected for good models, no non-glycine residues exhibit positive ~-angles, and about 85% of residues occupy the most favorable regions in Ramachandran space [15]. In comparison, a PROCHECK analysis on the NMR structure which was used as the starting point for the dynamics is shown in Fig. 5(b). Comparing the PROCHECK results of these two structures one observes that the average solution structure appears to be a better structure with 84% of the residues in most favored regions as opposed to 62% for the NMR structure. A comparison of the Chi-1 vs Chi-2 plot (Fig. 6) shows that the average solution structure has only 3 residues that are more than 2.5 standard deviations from ideal while the NMR structure has 10. In addition PROCHECK determined the secondary structure of the average solution structure which is consistent with the published NMR structure [2]. The average AFP solution structure has an RMS deviation from the NMR structure of 2.95 .~ for all non-hydrogen atoms, an RMS deviation of 2.37 A for backbone atoms (N, Ca, C, O), an RMS deviation of 2.31 A for all backbone atoms except those involving the 'loop' region (T28-L40), and an RMS of 2.56 for the backbone atoms involving only the 'loop' region. Before dynamics, the 'loop' region from T28 to L40 showed only partial helical character, however, the average dynamics structure shows the formation of a single-turn-helix comprised of residues 137, P38, R39 and L40. The average structure also reveals R48 which was partially buried in the NMR structure to be completely exposed to the solvent. In comparison, both the NMR and average solution structure display a fairly random distribution of polar and charged residues, the latter occurring in peripheral clusters with o

negative charges in close proximity to positive charges. In each case, a positively charged amino acid (R) has a negatively charged group (D or E) within a 6.7 A (Ca to Ca) range. The three arginines (23, 39 and 47) seem to be associated with E25, D36 and D58 respectively. The occurrence of a large number of ion pairs is similar to aldehyde ferrodoxin oxidoreductase and maybe necessary for protein stability [16]. K61 appears to be the exception by being very loosely associated with N46 The solvent accessible surface are of Type III AFP was found to be 57% apolar, 25% polar, and 17% charged, values which are comparable with those observed in a survey of water-soluble proteins [17]. Finally, PROCHECK identifies 32 protein-protein hydrogen bonds in the NMR structure versus 40 in the average solution structure. Although polar groups are common, neither structure offers a distinct plane of specifically spaced polar (or charged) amino acids typical of Type I AFPs. Polar residues do tend to lie on either side of the protein within and around each triple/~-sheet region, but the hydrophilic groups are generally interspersed among hydrophobic patches with little discernible pattern. o

3.2. Dynamics analysis

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The RMS deviation was calculated for the protein backbone atoms over the course of the simulation (see Fig. 7(a)). The RMS deviation for each successive data step was determined by comparing the current protein structure with the protein structure at the beginning of data collection phase. Over the final 30 ps of simulation, the average RMS deviation was 1.4 A. The radius of gyration, taken for the full 110 ps of data collection, oscillates around 11.1 A (Fig. 7(b)). The fluctuation around a constant value indicates that the structure is stabilized, although the RMS deviation from the initial structure is rising during the initial stages of the simulation. Fig. 8 is an RMS fluctuation plot of the Ca and C a atom of the individual residues o

Fig. 5. (a) The Ramachandran~/ff plotfromPROCHECKof the averagedstructurefromthe simulation.(b) The Ramachandran~/ffplot from PROCHECKof the initialNMR structure.The II's representnon-glyeineresiduesand the •'s representglycineresidues.As expectedfor good models, no non-glycineresiduesexhibitpositive~-angles, and about 85% of residues occupythe most favorableregionsin Ramachandran space.

J.D. Madura et al./Journal of Molecular Structure (Theochem) 388 (1996) 65-77

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Time(psi Fig. 7. (a) A plot of the RMS backbone deviation as a function of time. The structure at time 0 ps for the data collection phase is the structure used to compute the RMS for all of the other structures. (b) A plot of the radius of gyration as a function of time. over the 110 ps simulation. Regions that contain some degree of secondary structure have low RMS values while the other regions fluctuate more indicated by large RMS fluctuation values. An interesting feature of the RMS fluctuation plot (Fig. 8) is the periodicity in the fluctuations indicating that there are regions of low movement while there are some very flexible regions. This may be necessary to keep regions in contact with the ice rigid while maintaining protein flexibility upon binding to ice. 3.3. Hydrogen bonding

Protein-protein and protein-water hydrogen bonds were analyzed and are summarized in Fig. 9. Fig. 9(a) is a plot of the normalized number of protein-protein hydrogen bonds that occurred over the simulation

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Fig. 8. Plots of the RMS fluctuationfor the C, and Ca atoms of each of the amino acid residues. time. Fig. 9(b) shows the normalized number of protein-water hydrogen bonds present during the total simulation. Generally, protein-water interactions correlate well with the accessibility of the polar sidechain groups. The strongest hydrogen bonding was exhibited by the three positively charged arginines (R23, R39, R47), each displaying at least some intra-protein hydrogen bond interaction during the whole simulation. The negatively charged residues generally showed little hydrogen bonding with the exception of E25 which had a very strong intra-protein interaction and essentially no hydrogen bonding with the water. Taken with a relatively large intraprotein value for R23, the behavior of E25 seems to indicate a sustained salt-bridge association between the two residues as indicated earlier. The presence of both intra-protein and protein-water hydrogen bonding for the charged K61 and polar N46 along with their consistent proximity during dynamics

Fig. 6. (a) The Chi-1 vs. Chi-2 PROCHECK plot for the average structure. (b) The Chi-1 vs. Chi-2 PROCHECKplot for the NMR structure. These plots show the side chain conformation and indicate their deviation from normal values. Residues with deviations greater than 2~rare labeled.

74

J.D. Madura et al./Journal of Molecular Structure (Theochem) 388 (1996) 65-77 Protein - Protein Hydrogen Bond Analysis

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plots. The plotswereconstructedby countingthe numberof hydrogen bonds for each individual residue over the total simulation period and dividingthat value by the numberof snapshotsused. seem to reveal an association like the charge-charge coupling described above. Hydrogen bond analysis yielded no conclusive data concerning other possible charge-charge interactions. For polar groups, hydrogen bonding was variable, ranging from exclusive protein-water to general intra-protein interactions. The three conserved residues N14, T18, and Q44 are each hydrogen-bonded to at least one water during the entire simulation, and their solvent interactions are indistinguishable from most other polar residues such as N1, T28, or Q2. 3.4. Protein~ice modeling

As mentioned in the introduction, the amino acid sequences for the Type III AFPs are quite variable except for a few conserved residues that are common to all or most of the Type III proteins [3]. Due to the nature of the Type III AFP and the lack of ice-etching data we will focus our protein/ice modeling efforts on the interactions between the C-face of the Type III

AFP and the (100) face of ice. This specific interaction was chosen because of the three conserved residues (N14, T18, and Q44) which have been identified through mutation studies as being crucial for antifreeze activity [3]. These particular amino acids lead to a decrease in protein function when mutated to shorter polar amino acids. Being polar, these amino acids possess hydrogen bonding potential consistent with the binding residues of the Type I AFP. Fig. 4 shows three orthogonal views of the interactions between hydrophilic residues near the C-face of the protein interacting with the (100) ice plane. This face incorporated the three conserved residues observed in the mutation studies [3]. Fig. 4(a) shows a side view down the c-axis of ice. In this view one observes the three hydrophilic residues, drawn as ball-and-stick, hydrogen bonding to the ice surface. In Fig. 4(b), which is a 90° rotation of Fig. 4(a), one sees the hydrophobic portions of the three 'active' residues fitting into the 'groove' of the (100) ice surface. Finally Fig. 4(c) shows a top view, that is looking down the a-axis of ice with the c-axis running from top to bottom. This view shows the seemingly featureless nature of the (100) ice face as opposed to the (201) and (2-10) ice surfaces in which the Type I AFPs bind [7,9]. Fig. 10 shows the results from the binding energy surface scan. From this figure one observes there are several low energy regions which occur around (0.5, 1.0); the starting position, (3.5, 1.5), and (1, 5.5). These positions correspond closely to repeat translations along the surface and follows the 'lattice matching' hypothesis of the Type I binding mechanism.

4. Discussion 4.1. Dynamics

The dynamics simulation presented here reveals the Type III AFP to be heterogeneous in three dimensional structure as well as amino acid sequence with a wide distribution of polar and charged residues. The coordinates taken from a low-resolution NMR-structure change to some extent. In particular, the originally less-well defined loop changes conformation and regularizes into one helical turn between residues 3741. The presence of a helix in this region is consistent with the experimental data from NMR-spectroscopy,

J.D. Madura et aL/Journal of Molecular Structure (Theochem) 388 (1996) 65-77

75

Type III AFP on (100)ice Potential Energy Surface

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showing a otN(i,i + 3) cross peak from I37 to V41, several NN(i,i + 1) cross peaks and an upfield shift for the oLCH-resonances, all indicative for the presence of a helix. As a result of the length of this segment, and moreover, the presence of a proline in this region (P38) which lacks the amide hydrogen, the characteristic cross peak pattern for helices is interrupted and thus inconclusive in structure calculation based on NOE-data. The incorporation of a proline into a helical turn initially seemed unusual since the helical propensity for proline residues is low. However, it has been shown that the preferred position of proline in helices is the N + 1 cap position as in this case, where the lack of hydrogen bonding capability is not important and where the restriction of the backbone conformation supports the formation of the first turn [18].

The overall change between the NMR- and the dynamics structure leading to RMS deviation of 2.3 have their origin in the nature of the NMR-structure being the average of relatively low-resolution structures generated by a distance geometry procedure [2]. In addition to conformational imperfections caused by averaging, DG-structures are known to contain local VDW-overlaps and residual conformational strain. In such cases molecular dynamics calculations can be successfully used to refine the structure [19]. Running the simulation with the protein in solution also allowed us to obtain appropriate conformations of the amino acid side chains by preventing them from undergoing exaggerated interaction with the protein backbone which is typical of in vacuo simulations. Compared with the original

76

J.D. Madura et al./Journal of Molecular Structure (Theochem) 388 (1996) 65-77

NMR structure, polar and charged groups of the average structure generally have their side chains more extended and solvent accessible. Three relatively equidistant charge-charge clusters involving the arginines were observed for both the average solution and NMR structures. While R47 and in particular R39 still showed significant hydrogen bonding with water, R23 seems to form a permanent charge-charge bridge with E25. This interaction can be supported by NMR-spectroscopic observations. R23 eN and HeN chemical shifts are downfield shifted relative to the resonances of R39 and R47 and relative to expected values. Also, the non-degeneracy of R23 6CH- and E25 7CH proton resonances indicate a rotational restriction, which in the observed absence of steric reasons supports the proposed charge-charge interaction. Although not all charged residues show such a sustained interaction, these possible positive/ negative interactions seem to help maintain the integrity of the three dimensional structure by acting as stabilizing salt bridges. The pairing of most charges also adds to earlier observations. AFPs in general have a lower than average proportion of charged residues, and moreover, many AFP sequences have nearly equal number of positively a n d negatively charged groups [11]. The consequence can be expected to be a relative small and weak electrostatic potential around the protein. It is not clear how and if this affects the protein activity [1]. However as stated earlier the occurrence of a large number of ion pairs is similar to aldehyde ferrodoxin oxidoreductase and maybe necessary for protein stability [16]. 4.2. Protein-ice

As observed in winter flounder and shorthorn sculpin AFPs, binding of polar/charged groups tends to be specific within the pockets and grooves of the c- and a-axes [7-9]. The structural fit of these AFPs indicate a specific accommodation of the protein backbone and both hydrophilic and hydrophobic residues of the binding surface. Also it is possible that tetrahedral end groups of certain polar/charged residues bind ice by 'replacing' surface water molecules and assuming the tetrahedral hydrogen bonding arrangements within the ice lattice. While exact hydrogen bonding schemes cannot be established, pending the identification of the ice binding plane; it is clear from work

conducted on the Type I AFPs that the side chain length and orientation as well as the exact nature of side chain end groups (i.e. planar or tetrahedral, charged or polar, and steric properties) are very specific for certain directions along appropriate ice crystal planes. The same relationships are suggested by the activities of N14, T18, and Q44 in mutation studies and in the hydrogen bonding patterns in Fig. 9. From a steric perspective, there are three regions that may be conducive to binding to ice. The loophelical region encompasses several charged residues, while primarily polar groups are observed along the C- and N-faces. However, from mutagenesis experiments, it is the C-face which is the preferred region. Foremost, the results from this work seem to concur with the mutagenesis experiments, however, apart from this, the binding of the other AFP regions to ice cannot be completely ruled out. Another concern is that modeling using geometric and energetic criteria in a two-component docking approach may be inadequate. More adequate or complete information might be obtainable upon inclusion of solvation effects. This, however, requires the simulation of the protein at the complete ice-water interface, including the solvation in form of explicit surface water molecules in the calculations. Although such interface calculations are presently not feasible, recent progress might allow these simulations in the near future [20].

Acknowledgements We are grateful for financial support of this work from Research Corporation under Grant No. C-3121, NOAA-Sea Grant NA16RG0155, NSF/EPSCoR Grant No. EHR-9108761, and NSF Grant MCB9322602.

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J.D. Madura et al./Journal of Molecular Structure (Theochem) 388 (1996) 65-77

[5] C.C. Cheng, A.L. DeVries, in C.C. Cheng, A.L. DeVries (Eds), The Role of Antifreeze Glycopeptides and Pcptides in the Freezing Avoidance of Cold-Water Fish, Springer-Veflag, Berlin, 1991, 1-14. [6] P.W. Wilson, Cryoletters, 14 (1993) 31-36. [7] J.D. Madura, A. Wierzbicki, J.P. Harrington, R.H. Maughon, J.A. Raymond, C.SJ. Sikes, Amer. Chem. Soc., 116 (1994) 417-418. [8] D. Wen, R.A. Laursen, Biophys. J., 63 (1992) 1659-1662. [9] A. Wierzbicki, M.S, Taylor, C.A. Knight, J.D. Madura, J.P. Harrington, C.S. Sikcs, Biophys. L, 71 (1995) 8-18. [10] C.A. Knight, E. Dfiggers, A.L De Vries, Biophys. J. 64 (1993) 252-259. [11] F.D. S6nnichsen, B.D. Sykes, P.L. Davies, Protein Science, 4 (1995) 460-471. [12] W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.LJ. Klein, Chem. Phys., 79 (1983) 926-935. [13] Quanta/CHARMm 4.0 molecular modeling software from Molecular Simulations Inc. of San Diego, CA. Cerius 2

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molecular modeling software for materials research from Molecular Simulations Inc. of San Diego, CA. [14] B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan, M. Karplus, J. Comput. Chem., 4 (1983) 187-217. [15] R~,. Lakowski, M.W. MacArthur, D.S. Ross, J.M. Thornton, L Appl. Crysr., 26 (1993) 283-291. [16] M.K. Chan, S. Mukund, A. Kletzin, M.W.W. Adams, D.C. Rees, Science, 267 (1995) 1463-1469. [17] S. Miller, J. Janin, A.M. Lesk, C. Chothia, J. Mol. Biol., 196 (1987) 641. [18] LS. Richardson, D.C. Richardson, Science, 240 (1988) 16481652. [19] L. Chiche, C. Gaboriaud, A. Heitz, J.-P. Momon, B. Castro, P.A. Kollman, Proteins, 6 (1989) 405-417. [20] O.A. Karim, A.D.J. Haymet, J. Chem. Phys., 89 (1988) 68896896. [21] P. Kraulis, J. Appl. Cryst., 24 (1991) 946-950.