Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets

The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.     

Selenocysteine in proteins-properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Influence of hydration on the conformational stability and formation of bends in terminally blocked dipeptides

The conformations of 23 terminally blocked dipeptide sequences were examined by conformational energy calculations that included the effects of the aqueous solvent. Starting structures were derived from combinations of minimum-energy conformations of hydrated single residues. Their conformational energies were then minimized using the ECEPP potential (Empirical Conformational Energy Program for Peptides) with hydration included. Short-range interactions dominate in stabilizing the conformations of the hydrated dipeptides. Differences between conformational stabilities of hydrated and unhydrated dipeptides in many cases are due to the competition of solute–water and intramolecular hydrogen bonds. In other cases, perturbation of the hydration shell of the solute by close approach of solute atoms alters conformational preferences. Probabilities of formation of bends were calculated and compared to the corresponding quantities for unhydrated dipeptides and to those calculated from x-ray structures. For bends in dipeptides containing two nonpolar amino acids, computations omitting hydration yield better results. However, better agreement with experimental (x-ray) bend probabilities for dipeptides containing glycine or polar amino acids is obtained only in some sequences when hydration is included. The results are rationalized by the observation that, in proteins, bends containing nonpolar sequences occur on the inside, shielded from the solvent. Bends containing glycine or polar amino acids occur frequently on the surface of the protein, but they are not completely hydrated.     

Computational and mutational analysis of human glutaredoxin (thioltransferase): probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.     

Selenium in thioredoxin reductase: a mechanistic perspective

Most high M(r) thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys(1)-Cys(2)) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys(2) position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pK(a) as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pK(a) of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH.     

Molecular dynamics simulations and density functional theory studies of NALMA and NAGMA dipeptides

Classical molecular dynamics (MD) simulations using fixed charged force field (AMBER ff03) and density functional theory method using the M05-2X/6-31G^?? level of theory have been used to investigate the plasticity of the hydrogen bond formed between dipeptides of N-Acetyl-Leucine-MethylAmide (NALMA), N-Acetyl-Glycine-MethylAmide (NAGMA), and vicinity of water molecules at temperature of 300?K. We have noticed that 2–3 water molecules contribute to change in the conformations of dipeptides NAGMA and NALMA. The self-assembly of 11 water molecules leads to the formation of water bridge at vicinity of the dipeptides and it constrain the conformations of dipeptides. We have found that the energy balance between breaking of the C?=?O…H–N H bonds and the formation of the C?=?O…H–O (wat) H bonds may be one of the determining factors to control the dynamics of the folding process of protein molecules.     

Investigations of dipeptide structures containing pyrrolysine as N-terminal residues: a DFT study in gas and aqueous phase

A set of six dipeptides containing pyrrolysine invariably at their N-terminal positions is studied in gas and aqueous phase using a polarizable continuum model (PCM). The molecular geometries of the dipeptides are fully optimized at B3LYP/6-31++G(d,p) level of theory and a second derivative (frequency) analysis confirms that all the optimized geometries are true minima. The effects of solvation and identity of the varying C-terminal residue on the energetics, structural features of the peptide planes, values of the ψ and ? dihedrals, geometry around the α-carbon atoms and theoretically predicted vibrational spectra of the dipeptides are thoroughly analyzed. Solvation effects are found to modify the gas phase conformation of the dipeptides around ψ dihedrals while the identity of the varying C-terminal residue affect the values of ?, planarity of the peptide planes and geometry around the α-carbon atoms. The presence or absence of three types of intramolecular H-bonds, namely O…H–N, N…H–N and O…H–C that leave noticeable signatures in the IR spectra, play crucial roles in influencing the geometry of the peptide planes and in determining the energetics of the dipeptides.     

Determinants of cysteine pKa values in creatine kinase and alpha1-antitrypsin

The structural determinants of the unusually low pK(a) values of Cys282 in human creatine kinase and Cys232 in alpha1-antitrypsin were studied computationally. We have demonstrated that hydrogen bonding to the cysteine residue is the prime determinant for both proteins. In the case of creatine kinase, the hydrogen bond donors are a serine side chain and an amide NH-group, while in alpha1-antitrypsin the donor is an amide NH. Each hydrogen bond lowers the pK(a) by between 0.8 and 1.5 pH units. The 1.1-unit lowering due to the Ser284-Cys282 hydrogen bond is in good agreement with the 1.2-unit difference between the Cys282 pK(a) value of wild-type and the S284A mutant of creatine kinase.     

Selenocysteine in proteins—properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK_a of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of ⁷⁷Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase

Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK(a) values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)). In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.     

Interactions that favor the native over the non-native disulfide bond among residues 58-72 in the oxidative folding of bovine pancreatic ribonuclease A

In the initial stages of the oxidative folding of both bovine pancreatic ribonuclease A (RNase A) and a 58-72 fragment thereof from the fully reduced, denatured state, the 65-72 correctly paired disulfide bond forms in preponderance over the incorrectly paired 58-65 disulfide bond. Since both disulfide-bonded loops contain the same number of amino acid residues, the question arises as to whether the native pairing results from interactions within the 58-72 segment that lead to a nativelike structure even in its fully reduced form. To answer this question, the chain buildup procedure, based on ECEPP, including a solvation treatment, was used to generate the low-energy structures for the 58-72 RNase segment, beginning with residue 72 and building back to residue 58; in this fragment, all three Cys residues (at positions 58, 65, and 72) initially exist in the reduced (CysH) state. After the open-chain energy minima of the 65-72 peptide were generated, these conformations were allowed to form the 65-72 disulfide bond, and the energies of the resulting oxidized conformations were reminimized and rehydrated. The global minimum of the loop-closed 65-72 structure and many of the low-lying loop-closed minima could be superimposed on the energy-minimized X-ray structure for residues 65-72. The low-energy structures for the full open chain 58-72 peptide were then computed and were allowed to form disulfide bonds either between residues 65 and 72 (native) or between residues 58 and 65 (non-native), and their energies were reminimized and rehydrated in the loop-closed state. Although the overall fold of the 65-72 loop-closed global minimum was the same as for the energy-minimized X-ray structure of these residues, the overall rms deviation was 3.9 A because of local deviations among residues 58-64. In contrast, the 65-72 segment of the global minimum of the 58-72 fragment could be superimposed on the corresponding residues of the energy-minimized X-ray structure. The lowest-energy structure for the 58-65 non-native paired 58-72 sequence was 6 kcal/mol higher in energy than that for the 58-72 peptide with the 65-72 disulfide bond formed. These results suggest that the native pairing of the 65-72 peptide arises from energetic determinants (adoption of left-handed single-residue conformations by Gly 68, and side chain interactions involving Gln 69) contained within this peptide sequence.     

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.     

Evaluation of the conformational free energies of loops in proteins

In this paper we discuss the problem of including solvation free energies in evaluating the relative stabilities of loops in proteins. A conformational search based on a gas-phase potential function is used to generate a large number of trial conformations. As has been found previously, the energy minimization step in this process tends to pack charged and polar side chains against the protein surface, resulting in conformations which are unstable in the aqueous phase. Various solvation models can easily identify such structures. In order to provide a more severe test of solvation models, gas phase conformations were generated in which side chains were kept extended so as to maximize their interaction with the solvent. The free energies of these conformations were compared to that calculated for the crystal structure in three loops of the protein E. coli RNase H, with lengths of 7, 8, and 9 residues. Free energies were evaluated with a finite difference Poisson-Boltzmann (FDPB) calculation for electrostatics and a surface area-based term for nonpolar contributions. These were added to a gas-phase potential function. A free energy function based on atomic solvation parameters was also tested. Both functions were quite successful in selecting, based on a free energy criterion, conformations quite close to the crystal structure for two of the three loops. For one loop, which is involved in crystal contacts, conformations that are quite different from the crystal structure were also selected. A method to avoid precision problems associated with using the FDPB method to evaluate conformational free energies in proteins is described. © 1994 John Wiley & Sons, Inc.     

Amino acid side-chain partition energies and distribution of residues in soluble proteins

Energies required to transfer amino acid side chains from water to less polar environments were calculated from results of several studies and compared with several statistical analyses of residue distributions in soluble proteins. An analysis that divides proteins into layers parallel with their surfaces is more informative than those that simply classify residues as exposed or buried. Most residues appear to be distributed as a function of the distance from the protein-water interface in a manner consistent with partition energies calculated from partitioning of amino acids between water and octanol phases and from solubilities of amino acids in water, ethanol, and methanol. Lys, Arg, Tyr, and Trp residues tend to concentrate near the water-protein interface where their apolar side-chain components are more buried than their polar side-chain components. Residue distributions calculated in this manner do not correlate well with side-chain solvation energies calculated from vapor pressures of side-chain analogs over a water phase. Results of statistical studies that classify residues as exposed to solvent or buried inside the protein interior appear to depend on the method used to classify residues. Data from some of these studies correlate better with solvation energies, but other data correlate better with partition energies. Most other statistical methods that have been used to evaluate effects of water on residue distributions yield results that correlate better with partition energies than with solvation energies.     

Conformational preferences of 4-chloroproline residues

Conformational preferences of the (2S,4R)-4-chloroproline (Clp) and (2S,4S)-4-chloroproline (clp) residues are explored at the M06-2X/cc-pVTZ//M06-2X/6-31+G(d) level of theory in the gas phase and in water, where solvation free energies were calculated using the implicit solvation model, and by an X-ray diffraction study in the solid state. In the gas phase, the down-puckered γ-turn structure with the trans prolyl peptide bond is most preferred for both Ac-Clp-NHMe and Ac-clp-NHMe, in which the C(7) hydrogen bond between two terminal groups seems to play a role, as found for Ac-Pro-NHMe. In water, the Clp residue has a strong preference for the up-puckered PP(II) structure, whereas the up-puckered PP(II) structure prevails a little over the down-puckered PP(II) structure for the clp residue, similar to the Pro residue. Hence, our calculated results on the puckering preference of the Clp and clp residues in water are in accord with the observed results deduced from the relative stabilities of the triple helices of the collagen model peptides. The X-ray structure of Ac-clp-NHMe was found to be the most preferred in water but that of Ac-Clp-NHMe was located as a local minimum with ΔG = 2.0 kcal/mol. In particular, the X-ray structure of Ac-Clp-NHMe was quite different from that of Ac-Clp-OMe but similar to that of Ac-Pro-NHMe. The lowest rotational barriers to the prolyl cis-trans isomerization for Ac-Clp-NHMe become nearly the same as those for Ac-Pro-NHMe in water, whereas the barriers are lower by ～2 kcal/mol for Ac-clp-NHMe. It was found that the cis-trans isomerization may proceed through the clockwise or anticlockwise rotations for Ac-Clp-NHMe and the anticlockwise rotation for Ac-clp-NHMe and Ac-Pro-NHMe in water.     

Structure of selenophosphate synthetase essential for selenium incorporation into proteins and RNAs

Selenophosphate synthetase (SPS) catalyzes the activation of selenide with adenosine 5'-triphosphate (ATP) to generate selenophosphate, the essential reactive selenium donor for the formation of selenocysteine (Sec) and 2-selenouridine residues in proteins and RNAs, respectively. Many SPS are themselves Sec-containing proteins, in which Sec replaces Cys in the catalytically essential position (Sec/Cys). We solved the crystal structures of Aquifex aeolicus SPS and its complex with adenosine 5'-(alpha,beta-methylene) triphosphate (AMPCPP). The ATP-binding site is formed at the subunit interface of the homodimer. Four Asp residues coordinate four metal ions to bind the phosphate groups of AMPCPP. In the free SPS structure, the two loop regions in the ATP-binding site are not ordered, and no enzyme-associated metal is observed. This suggests that ATP binding, metal binding, and the formation of their binding sites are interdependent. To identify the amino-acid residues that contribute to SPS activity, we prepared six mutants of SPS and examined their selenide-dependent ATP consumption. Mutational analyses revealed that Sec/Cys13 and Lys16 are essential. In SPS.AMPCPP, the N-terminal loop, including the two residues, assumes different conformations ("open" and "closed") between the two subunits. The AMPCPP gamma-phosphate group is solvent-accessible, suggesting that a putative nucleophile could attack the ATP gamma-phosphate group to generate selenophosphate and adenosine 5'-diphosphate (ADP). Selenide attached to Sec/Cys13 as -Se-Se(-)/-S-Se(-) could serve as the nucleophile in the "closed" conformation. A water molecule, fixed close to the beta-phosphate group, could function as the nucleophile in subsequent ADP hydrolysis to orthophosphate and adenosine 5'-monophosphate.     

The role of carbon-donor hydrogen bonds in stabilizing tryptophan conformations

Although most side-chain torsion angles correspond to low-energy rotameric positions, deviations occur with significant frequency. One striking example arises in Trp residues, which have an important role in stabilizing protein structures because of their size and mixed hydrophobic/hydrophilic character. Ten percent of Trp side-chains have unexplained conformations with chi(2) near 0 degrees instead of the expected 90 degrees. The current work is a structural and energetic analysis of these conformations. It is shown that many Trp residues with these orientations are stabilized by three-center carbon-donor hydrogen bonds of the form C-H...X...H-C, where X is a polar hydrogen-bond acceptor in the environment of the side-chain. The bridging hydrogen bonds occur both within the Trp side-chain and between the side-chain and the local protein backbone. Free energy maps of an isolated Trp residue in an explicit water environment show a minimum corresponding to the off-rotamer peak observed in the crystallographic data. Bridging carbon-donor hydrogen bonds are also shown to stabilize on-rotamer Trp conformations, and similar bridging hydrogen bonds also stabilize some off-rotamer Asp conformations. The present results suggest a previously unrecognized role for three-center carbon-donor hydrogen bonds in protein structures and support the view that the off-rotamer Trp side-chain orientations are real rather than artifacts of crystallographic refinements. Certain of the off-rotamer Trp conformations appear to have a functional role.     

Conformational preferences and prolyl cis–trans isomerization of phosphorylated Ser/Thr‐Pro motifs

The conformational study on Ac‐pSer‐Pro‐NHMe and Ac‐pThr‐Pro‐NHMe peptides has been carried out using hybrid density functional methods with the implicit solvation reaction field theory at the B3LYP/ 6‐311++G(d,p)//B3LYP/6‐31+G(d) level of theory in the gas phase and in solution (chloroform and water). For both pSer‐Pro and pThr‐Pro peptides in the gas phase and in chloroform, the most preferred conformation has the α‐helical structure for the pSer/pThr residue, the down‐puckered polyproline I structure for the Pro residue, and the cis prolyl peptide bond between the two residues, in which two hydrogen bonds between the phosphate oxygens with the backbone N? H groups seem to play a role. However, the trans conformations that have a single hydrogen bond of the phosphate oxygen with either of two backbone N? H groups become most preferred for both peptides in water. This is because the hydration free energy of the anionic oxygen of the phosphate group is expected to dramatically decrease for the cis conformation upon formation of the hydrogen bond with the backbone N? H groups. These calculated results are consistent with the observations by NMR and IR experiments, suggesting the existence of hydrogen bonds between the charged phosphoryl group and the backbone amide protons in solution. The calculated cis populations of 14.7 and 14.2% and rotational barriers of 19.87 and 20.57 kcal/mol to the cis‐to‐trans isomerization for pSer‐Pro and pThr‐Pro peptides in water, respectively, are consistent with the observed values for pSer‐Pro and pThr‐Pro containing peptides from NMR experiments. However, the hydrogen bond between the prolyl nitrogen and the following amide N? H group, which was suggested to be capable of catalyzing the prolyl isomerization, does not play a role in stabilizing the preferred transition state for the pSer/pThr‐Pro peptides in water. Instead, the amide hydrogen of the NHMe group is involved in a bifurcated hydrogen bond with the anionic oxygen and phosphoester oxygen of the phosphate group. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 330–339, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hydrogen bonding is the prime determinant of carboxyl pKa values at the N-termini of alpha-helices

Experimentally determined mean pK(a) values of carboxyl residues located at the N-termini of alpha-helices are lower than their overall mean values. Here, we perform three types of analyses to account for this phenomenon. We estimate the magnitude of the helix macrodipole to determine its potential role in lowering carboxyl pK(a) values at the N-termini. No correlation between the magnitude of the macrodipole and the pK(a) values is observed. Using the pK(a) program propKa we compare the molecular surroundings of 18 N-termini carboxyl residues versus 233 protein carboxyl groups from a previously studied database. Although pK(a) lowering interactions at the N-termini are similar in nature to those encountered in other protein regions, pK(a) lowering backbone and side-chain hydrogen bonds appear in greater number at the N-termini. For both Asp and Glu, there are about 0.5 more hydrogen bonds per residue at the N-termini than in other protein regions, which can be used to explain their lower than average pK(a) values. Using a QM-based pK(a) prediction model, we investigate the chemical environment of the two lowest Asp and the two lowest Glu pK(a) values at the N-termini so as to quantify the effect of various pK(a) determinants. We show that local interactions suffice to account for the acidity of carboxyl residues at the N-termini. The effect of the helix dipole on carboxyl pK(a) values, if any, is marginal. Backbone amide hydrogen bonds constitute the single biggest contributor to the lowest carboxyl pK(a) values at the N-termini. Their estimated pK(a) lowering effects range from about 1.0 to 1.9 pK(a) units.     

Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: comparison with functionally related proteins.

The chemistry of active-site cysteine residues is central to the activity of thiol-disulfide oxidoreductases of the thioredoxin superfamily. In these reactions, a nucleophilic thiolate is required, but the associated pK(a) values differ vastly in the superfamily, from less than 4 in DsbA to greater than 7 in Trx. The factors that stabilize this thiolate are, however, not clearly established. The glutaredoxins (Grxs), which are members of this superfamily, contain a Cys-Pro-Tyr-Cys motif in their active site. In reduced Grxs, the pK(a) of the N-terminal active-site nucleophilic cysteine residue is lowered significantly, and the stabilization of the corresponding thiolate is expected to influence the redox potential of these enzymes. Here, we use a combination of long molecular dynamics (MD) simulations, pK(a) calculations, and experimental investigations to derive the structure and dynamics of the reduced active site from Escherichia coli Grx3, and investigate the factors that stabilize the thiolate. Several different MD simulations converged toward a consensus conformation for the active-site cysteine residues (Cys11 and Cys14), after a number of local conformational changes. Key features of the model were tested experimentally by measurement of NMR scalar coupling constants, and determination of pK(a) values of selected residues. The pK(a) values of the Grx3 active-site residues were calculated during the MD simulations, and support the underlying structural model. The structure of Grx3, in combination with the pK(a) calculations, indicate that the pK(a) of the N-terminal active-site cysteine residue in Grx3 is intermediate between that of its counterpart in DsbA and Trx. The pK(a) values in best agreement with experiment are obtained with a low (<4) protein dielectric constant. The calculated pK(a) values fluctuate significantly in response to protein dynamics, which underscores the importance of the details of the underlying structures when calculating pK(a) values. The thiolate of Cys11 is stabilized primarily by direct hydrogen bonding with the amide protons of Tyr13 and Cys14 and the thiol proton of Cys14, rather than by long-range interactions from charged groups or from a helix macrodipole. From the comparison of reduced Grx3 with other members of the thioredoxin superfamily, a unifying theme for the structural basis of thiol pK(a) differences in this superfamily begins to emerge.