Thermal folding and mechanical unfolding pathways of protein secondary structures

Mechanical stretching of secondary structures is studied through molecular dynamics simulations of a Go-like model. Force versus displacement curves are studied as a function of the stiffness and velocity of the pulling device. The succession of stretching events, as measured by the order in which contacts are ruptured, is compared to the sequencing of events during thermal folding and unfolding. Opposite cross-correlations are found for an alpha-helix and a beta-hairpin structure. In a tandem of two alpha-helices, the two constituent helices unravel nearly simultaneously. A simple condition for simultaneous versus sequential unraveling of repeat units is presented.     

Secondary structure length as a determinant of folding rate of proteins with two- and three-state kinetics

We present a simple method for determining the folding rates of two- and three-state proteins from the number of residues in their secondary structures (secondary structure length). The method is based on the hypothesis that two- and three-state foldings share a common pattern. Three-state proteins first condense into metastable intermediates, subsequent forming of alpha-helices, turns, and beta-sheets at slow rate-limiting step. The folding rate of such proteins anticorrelate with the length of these beta-secondary structures. It is also assumed that in two-state folding, rapidly folded alpha-helices and turns may facilitate formation of fleeting unobservable intermediates and thus show two-state behavior. There is an inverse relationship between the folding rate and the length of beta-sheets and loops. Our study achieves 94.0 and 88.1% correlations with folding rates determined experimentally for 21 three- and 38 two-state proteins, respectively, suggesting that protein-folding rates are determined by the secondary structure length. The kinetic kinds are selected on the basis of a competitive formation of hydrophobic collapse and alpha-structure in early intermediates.     

Contribution of secondary structure to the heat capacity and enthalpy distribution of the unfolded state in proteins.

We have recently shown that one can construct the enthalpy distribution for protein molecules from experimental knowledge of the temperature dependence of the heat capacity. For many proteins the enthalpy distribution evaluated at the midpoint of the denaturation transition (corresponding to the maximum in the heat capacity vs temperature curve) is broad and biphasic, indicating two different populations of molecules (native and unfolded) with distinctly different enthalpies. At temperatures above the denaturation point, the heat capacity for the unfolded state in many proteins is quite large and using the analysis just mentioned, we obtain a gaussian-like enthalpy distribution that is very broad. A large value of the heat capacity indicates that there are structural changes going on in the unfolded state above the transition temperature. In the present paper we investigate the origin of this large heat capacity by considering the presence of changing amounts of secondary structure (specifically, alpha-helix) in the unfolded state. For this purpose we use the empirical estimates of the Zimm-Bragg sigma and s factors for all of the native amino acids in water as determined by Scheraga and co-workers. Using myoglobin as an example, we calculate probability profiles and distribution functions for the total number of helix states in the specific-sequence molecule. Given the partition function for the specific-sequence molecule, we can then calculate a set of enthalpy moments for the molecule from which we obtain a good estimate of the enthalpy distribution in the unfolded state. This distribution turns out to be quite narrow when compared with the distribution obtained from the raw heat capacity data. We conclude that there must be other major structural changes (backbone and solvent) that are not accounted for by the inclusion of alpha-helix in the unfolded state.     

CD studies on films of amyloid proteins and polypeptides: quantitative g-factor analysis indicates a common folding motif

Irrespective of the constituent protein, all amyloid fibrils show similar morphology in the electron microscope and x-ray diffraction patterns characteristic of a "cross-beta" structure, with extended beta-strands perpendicular to the fibril's long axis. Little is known about the amount or type of this structure. I have measured CD spectra of films formed from a number of amyloid proteins and polypeptides, and estimated their contents of extended secondary structure, by analysis of their g-factor spectra, the ratio of the CD and absorbance signals (P. McPhie, Analytical Biochemistry, 2001, Vol. 293, pp. 109-119). Amyloid films of Abeta-(1-40) peptide, beta-2-microglobulin, insulin, and three homopolypeptides show very intense CD spectra, compatible with the presence of a beta-helix-like structure, arranged in a common framework in the fibrils. The extent of this structure was estimated as 45-80% in the protein fibrils and 30-80% in the polypeptide fibrils.     

Prediction of protein secondary structures from conformational biases

We use LINUS (the "Local Independently Nucleated Units of Structure"), a procedure developed by Srinivasan and Rose, to provide a physical interpretation of and predict the secondary structures of proteins. The secondary structure type at a given site is identified by the largest conformational bias during short simulations. We examine the rate of successful prediction as a function of temperature and the interaction window. At high temperatures, there is a large propensity for the establishment of beta-strands whereas alpha-helices appear only when the temperature is lower than a certain threshold value. It is found that there exists an optimal temperature at which the correct secondary structures are predicted most accurately. We find that this temperature is close to the peak temperature of the specific heat. Changing the interaction window or carrying out longer simulations approaching equilibrium lead to little change in the optimal success rate. Our findings are in accord with the observation by Srinivasan and Rose that the secondary structures are mainly determined by local interactions and appear in the early stage of folding.     

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of α-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining α-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The α-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each α-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set I] and [set 2]) and for 30 mostly α-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix—coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix—coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area. © 1997 John Wiley & Sons, Inc. Biopoly 42: 239–269, 1997     

Cooperative effects in hydrogen-bonding of protein secondary structure elements: a systematic analysis of crystal data using Secbase

A systematic analysis of the hydrogen-bonding geometry in helices and beta sheets has been performed. The distances and angles between the backbone carbonyl O and amide N atoms were correlated considering more than 1500 protein chains in crystal structures determined to a resolution better than 1.5 A. They reveal statistically significant trends in the H-bond geometry across the different secondary structural elements. The analysis has been performed using Secbase, a modular extension of Relibase (Receptor Ligand Database) which integrates information about secondary structural elements assigned to individual protein structures with the various search facilities implemented into Relibase. A comparison of the mean hydrogen-bond distances in alpha helices and 3(10) helices of increasing length shows opposing trends. Whereas in alpha helices the mean H-bond distance shrinks with increasing helix length and turn number, the corresponding mean dimension in 3(10) helices expands in a comparable series. Comparing similarly the hydrogen-bond lengths in beta sheets there is no difference to be found between the mean H-bond length in antiparallel and parallel beta sheets along the strand direction. In contrast, an interesting systematic trend appears to be given for the hydrogen bonds perpendicular to the strands bridging across an extended sheet. With increasing number of accumulated strands, which results in a growing number of back-to-back piling hydrogen bonds across the strands, a slight decrease of the mean H-bond distance is apparent in parallel beta sheets whereas such trends are obviously not given in antiparallel beta sheets. This observation suggests that cooperative effects mutually polarizing spatially well-aligned hydrogen bonds are present either in alpha helices and parallel beta sheets whereas such influences seem to be lacking in 3(10) helices and antiparallel beta sheets.     

The distribution of alpha-helix propensity along the polypeptide chain is not conserved in proteins from the same family.

We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of alpha-helices. We have analyzed by CD the conformation of peptides corresponding to the five alpha-helices of two alpha/beta parallel proteins (ComA and Ara). The large alpha-helical population of peptide ComA-4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21-ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275-296). Comparison of the helical content of equivalent peptides reveals that protein alpha-helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.     

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.     

Structures of intact glycoproteins from vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra for the glycoproteins alpha1-acid glycoprotein (AGP) and bovine submaxillary mucin (BSM), have been measured in D2O solutions and for the films prepared from aqueous (H2O) buffer solutions in the 1800 to 900 cm(-1) region. The solution VCD results revealed that AGP has beta-sheet structure, along with a significant amount of alpha-helix as evidenced from a W pattern in the amide I region. The VCD of BSM solution suggested a polyproline II type structure, characterized by the appearance of strong negative couplet in the amide I region. The film VCD results on AGP and BSM suggested that the secondary structures of polypeptide fold in the film state are similar to those in the solution. The absence of any significant film VCD in the low frequency region (1200-900 cm(-1)), suggested that the dominant linkage for carbohydrate residues is likely to be a beta linkage. VCD spectroscopy gains importance in the secondary structural analysis of polypeptide fold in glycoproteins due to the absence of interfering VCD from the carbohydrate residues in the conformationally sensitive amide I region. Also, film VCD studies permit measurements in the low wavenumber region (1200-900 cm(-1)) that reveal the dominant type of linkage for carbohydrate residues. Such clear structural information is unlike that from ECD, where ECD bands of acylated amino sugar residues interfere with those of polypeptide backbone in the conformationally sensitive far-UV region.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

Prediction of secondary structural content of proteins from their amino acid composition alone. I. New analytic vector decomposition methods

The predictive limits of the amino acid composition for the secondary structural content (percentage of residues in the secondary structural states helix, sheet, and coil) in proteins are assessed quantitatively. For the first time, techniques for prediction of secondary structural content are presented which rely on the amino acid composition as the only information on the query protein. In our first method, the amino acid composition of an unknown protein is represented by the best (in a least square sense) linear combination of the characteristic amino acid compositions of the three secondary structural types computed from a learning set of tertiary structures. The second technique is a generalization of the first one and takes into account also possible compositional couplings between any two sorts of amino acids. Its mathematical formulation results in an eigenvalue/eigenvector problem of the second moment matrix describing the amino acid compositional fluctuations of secondary structural types in various proteins of a learning set. Possible correlations of the principal directions of the eigenspaces with physical properties of the amino acids were also checked. For example, the first two eigenvectors of the helical eigenspace correlate with the size and hydrophobicity of the residue types respectively. As learning and test sets of tertiary structures, we utilized representative, automatically generated subsets of Protein Data Bank (PDB) consisting of non-homologous protein structures at the resolution thresholds ≤1.8Å, ≤2.0Å, ≤2.5Å, and ≤3.0Å. We show that the consideration of compositional couplings improves prediction accuracy, albeit not dramatically. Whereas in the self-consistency test (learning with the protein to be predicted), a clear decrease of prediction accuracy with worsening resolution is observed, the jackknife test (leave the predicted protein out) yielded best results for the largest dataset (≤3.0 Å, almost no difference to the self-consistency test!), i.e., only this set, with more than 400 proteins, is sufficient for stable computation of the parameters in the prediction function of the second method. The average absolute error in predicting the fraction of helix, sheet, and coil from amino acid composition of the query protein are 13.7, 12.6, and 11.4%, respectively with r.m.s. deviations in the range of 8.6 ÷ 11.8% for the 3.0 Å dataset in a jackknife test. The absolute precision of the average absolute errors is in the range of 1 ÷ 3% as measured for other representative subsets of the PDB. Secondary structural content prediction methods found in the literature have been clustered in accordance with their prediction accuracies. To our surprise, much more complex secondary structure prediction methods utilized for the same purpose of secondary structural content prediction achieve prediction accuracies very similar to those of the present analytic techniques, implying that all the information beyond the amino acid composition is, in fact, mainly utilized for positioning the secondary structural state in the sequence but not for determination of the overall number of residues in a secondary structural type. This result implies that higher prediction accuracies cannot be achieved relying solely on the amino acid composition of an unknown query protein as prediction input. Our prediction program SSCP has been made available as a World Wide Web and E-mail service. © 1996 Wiley-Liss, Inc.     

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.     

Folding of beta/alpha-unit scrambled forms of S. cerevisiae triosephosphate isomerase: Evidence for autonomy of substructure formation and plasticity of hydrophobic and hydrogen bonding interactions in core of (beta/alpha)8-barrel

We show that residues at the interfaces of protein-protein complexes have higher side-chain energy than other surface residues. Eight different sets of protein complexes were analyzed. For each protein pair, the complex structure was used to identify the interface residues in the unbound monomer structures. Side-chain energy was calculated for each surface residue in the unbound monomer using our previously developed scoring function.1 The mean energy was calculated for the interface residues and the other surface residues. In 15 of the 16 monomers, the mean energy of the interface residues was higher than that of other surface residues. By decomposing the scoring function, we found that the energy term of the buried surface area of non-hydrogen-bonded hydrophilic atoms is the most important factor contributing to the high energy of the interface regions. In spite of lacking hydrophilic residues, the interface regions were found to be rich in buried non-hydrogen-bonded hydrophilic atoms. Although the calculation results could be affected by the inaccuracy of the scoring function, patch analysis of side-chain energy on the surface of an isolated protein may be helpful in identifying the possible protein-protein interface. A patch was defined as 20 residues surrounding the central residue on the protein surface, and patch energy was calculated as the mean value of the side-chain energy of all residues in the patch. In 12 of the studied monomers, the patch with the highest energy overlaps with the observed interface. The results are more remarkable when only three residues with the highest energy in a patch are averaged to derive the patch energy. All three highest-energy residues of the top energy patch belong to interfacial residues in four of the eight small protomers. We also found that the residue with the highest energy score on the surface of a small protomer is very possibly the key interaction residue.     

Conformational change from antiparallel beta-sheet to alpha-helix in a series of depsipeptide, -(Leu-Leu-Lac)(n)-: syntheses, spectroscopic studies, and crystal structures of Boc-Leu-Lac-OEt and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2)

The depsipeptides Boc-Leu-Lac-OEt (1) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2) (2 and 3, respectively) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized and studied by crystallographic, CD spectroscopic, and ESI-MS analyses. In the packing cells, those three compounds adopt beta-strand conformations. Each molecule is linked into a dimer (1) or an infinite assembly (2 and 3) by tight hydrogen bonds of the type NH...O==C. Interestingly, the hexamer, 3 shows the first example of antiparallel pleated beta-sheet crystal structure for a depsipeptide molecule. In the packing cells, especially for 3, the ester groups O--C==O are perpendicularly oriented to the amide groups NH--C==O and beta-sheet planes to avoid the interaction between --O--(ester) and O==C. Therefore, when the chain length become longer, the O...O==C repulsion interaction works as a beta-sheet breaker and hence promotes an alpha-helical structure as observed for Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (4) (Oku et al. Biopolymers 2004, 75, 242-254) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 4-6) (5-7) (Katakai et al., Biopolymers 1996, 38, 285-290), in which the O...O==C repulsion does not cause significant structural changes in alpha-helical main chains. Therefore from the structural and spectroscopic analyses, we have found governing factors for the specificity in the beta-sheet and alpha-helix decision in this series of depsipeptides, -(Leu-Leu-Lac)(n)-.     

Addition of a peptide fragment on an alpha-helical depsipeptide induces alpha/3(10)-conjugated helix: synthesis, crystal structure, and CD spectra of Boc-Leu-Leu-Ala-(Leu-Leu-Lac)3-Leu-Leu-OEt

The depsipeptide Boc(1)-Leu(2)-Leu(3)-Ala(4)-Leu(5)-Leu(6)-Lac(7)-Leu(8)-Leu(9)-Lac(10)-Leu(11)-Leu(12)-Lac(13)-Leu(14)-Leu(15)-OEt(16) (1) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized from the peptide Boc-Leu-Leu-Ala-OEt (2) and a depsipeptide, Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (3). Single crystals of 1 were successfully obtained and the structure has been solved by direct methods (such as Sir2002 and Shake-and-Bake). Interestingly, 1 adopts an alpha/3(10)-conjugated helix containing a kink at the junction of peptide and depsipeptide segments, Leu3-Lac7. This is significantly different from the conformation of 3, which has a straight alpha-helical structure with standard phi and psi angles. Microcrystalline CD spectra were also studied to compare structural properties of 1 and 3. The differences between alpha/3(10)- and alpha-helices appear in these CD spectra.     

The origins of asymmetry in the folding transition states of protein L and protein G

Topology has been shown to be an important determinant of many features of protein folding; however, the delineation of sequence effects on folding remains obscure. Furthermore, differentiation between the two influences proves difficult due to their intimate relationship. To investigate the effect of sequence in the absence of significant topological differences, we examined the folding mechanisms of segment B1 peptostreptococcal protein L and segment B1 of streptococcal protein G. These proteins share the same highly symmetrical topology. Despite this symmetry, neither protein folds through a symmetrical transition state. We analyzed the origins of this difference using theoretical models. We found that the strength of the interactions present in the N-terminal hairpin of protein L causes this hairpin to form ahead of the C-terminal hairpin. The difference in chain entropy associated with the formation of the hairpins of protein G proves sufficient to beget initiation of folding at the shorter C-terminal hairpin. Our findings suggest that the mechanism of folding may be understood by examination of the free energy associated with the formation of partially folded microstates.     

Formation of oriented polypeptides on Au(111) surface depends on the secondary structure controlled by peptide length.

We synthesized three different lengths of poly(L-lysine) containing an -SH group at the terminal (PLL(n)-SH, n (polymerization degree) = 4, 10, 30) and adsorbed them on an Au(111) surface. To analyze the formation process and the structure of self-assembled monolayers (SAMs), we used atomic force microscopy (AFM) and Fourier transform infrared reflection absorption spectra (FT-IR RAS). At the initial stage of SAM growth, formation of nanosize domains was confirmed by AFM imaging. The alpha-helical PLL(30)-SH exhibited a well-defined SAM structure after adsorption reached equilibrium. The alpha-helical PLL(30)-SH was almost perpendicular to the gold surface and exhibited interesting molecular packing due to the secondary structure of PLL(30)-SH and the underlying Au(111) array. The tilt angle of the helix axis from the substrate normal was estimated to be about 50 degrees (AFM) and 44 degrees (FT-IR RAS) respectively. On the other hand, PLL(4)-SH and PLL(10)-SH formed beta-sheet-type SAMs on the Au(111) surface based on the structure determined by FT-IR RAS spectrum.     

Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18

The three ribosomal proteins L7, S5, and S18 are included in the rare subset of prokaryotic proteins that are known to be N(alpha)-acetylated. The GCN5-related N-acetyltransferase (GNAT) protein RimI, responsible for the N(alpha)-acetylation of the ribosomal protein S18, was cloned from Salmonella typhimurium LT2 (RimI(ST)), overexpressed, and purified to homogeneity. Steady-state kinetic parameters for RimI(ST) were determined for AcCoA and a peptide substrate consisting of the first six amino acids of the target protein S18. The crystal structure of RimI(ST) was determined in complex with CoA, AcCoA, and a CoA-S-acetyl-ARYFRR bisubstrate inhibitor. The structures are consistent with a direct nucleophilic addition-elimination mechanism with Glu103 and Tyr115 acting as the catalytic base and acid, respectively. The RimI(ST)-bisubstrate complex suggests that several residues change conformation upon interacting with the N terminus of S18, including Glu103, the proposed active site base, facilitating proton exchange and catalysis.