Coordination topology and stability for the native and binding conformers of chymotrypsin inhibitor 2.

We demonstrate that the stabilization of the binding region is accomplished at the expense of a loss in the stability of the rest of the protein. A novel molecular mechanics (MM) approach is introduced to distinguish residue stabilities of proteins in a given conformation. As an example, the relative stabilities of folded chymotrypsin inhibitor 2 (CI2) in unbound form, and CI2 in complex with subtilisin novo is investigated. The conformation of the molecule in the two states is almost identical, with an approximately 0.6-A root-mean-square deviation (RMSD) of the Calpha atoms. On binding, the packing density changes only at the binding loop. However, residue fluctuations in the rest of the protein are greatly altered solely due to those contacts, indicating the effective propagation of perturbation and the presence of remotely controlling residues. To quantify the interplay between packing density, packing order, residue fluctuations, and residue stability, we adopt an MM approach whereby small displacements are inserted at selected residues, followed by energy minimization; the displacement of each residue in response to such perturbations are organized in a perturbation-response matrix L. We define residue stability lambda(i) = summation operator((j)L(ij))/ summation operator((j) L(ji)) as the ratio of the amount of change to which the residue is amenable, to the ability of a given residue to induce change. We then define the free energy associated with residue stability, DeltaG(lambda) = -RT ln lambda. DeltaG(lambda) intrinsically selects the residues that are in the folding core. Upon complexation, the binding loop becomes more resistant to perturbation, in contrast to the alpha-helix that favors change. Although the two forms of CI2 are structurally similar, residue fluctuations differ vastly, and the stability of many residues is altered upon binding. The decrease in entropy introduced by binding is thus compensated by these changes.

Elucidating the structural mechanisms for biological activity of the chemokine family.

Chemokines are a family of proteins involved in inflammatory and immune response. They share a common fold, made up of a three-stranded beta-sheet, and an overlaying alpha-helix. Chemokines are mainly categorized into two subfamilies distinguished by the presence or absence of a residue between two conserved cysteines in the N-terminus. Although dimers and higher-order quaternary structures are common in chemokines, they are known to function as monomers. Yet, there is quite a bit of controversy on how the actual function takes place. The mechanisms of binding and activation in the chemokine family are investigated using the gaussian network model of proteins, a low-resolution model that monitors the collective motions in proteins. It is particularly suitable for elucidating the global dynamic characteristics of large proteins or the common properties of a group of related proteins such as the chemokine family presently investigated. A sample of 16 proteins that belong to the CC, CXC, or CX(3)C subfamilies are inspected. Local packing density and packing order of residues are used to determine the type and range of motions on a global scale, such as those occurring between various loop regions. The 30s-loop, although not directly involved in the binding interface like the N-terminus and the N-loop, is identified as having a prominent role in both binding/activation and dimerization. Two mechanisms are distinguished based on the communication among the three flexible regions. In these two-step mechanisms, the 30s-loop assists either the N-loop or the N-terminus during binding and activation. The findings are verified by molecular mechanics and molecular dynamics simulations carried out on the detailed structure of representative proteins from each mechanism type. A basis for the construction of hybrids of chemokines to bind and/or activate various chemokine receptors is presented. Proteins 2001;43:150-160.

On the transferability of atomic solvation parameters: Ab initio structural prediction of cyclic heptapeptides in DMSO.

A statistical mechanics methodology for predicting the solution structures and populations of peptides developed recently is based on a novel method for optimizing implicit solvation models, which was applied initially to a cyclic hexapeptide in DMSO (C. Baysal and H. Meirovitch, Journal of American Chemical Society, 1998, vol. 120, pp. 800-812). Thus, the molecule has been described by the simplified energy function E(tot) = E(GRO) + summation operator(k) sigma(k)A(k), where E(GRO) is the GROMOS force-field energy, sigma(k) and A(k) are the atomic solvation parameter (ASP) and the solvent accessible surface area of atom k, respectively. In a more recent study, these ASPs have been found to be transferable to the cyclic pentapeptide cyclo(D-Pro(1)-Ala(2)-Ala(3)-Ala(4)-Ala(5)) in DMSO (C. Baysal and H. Meirovitch, Biopolymers, 2000, vol. 53, pp. 423-433). In the present paper, our methodology is applied to the cyclic heptapeptides axinastatin 2 [cyclo(Asn(1)-Pro(2)-Phe(3)-Val(4)-Leu(5)-Pro(6)-Val(7))] and axinastatin 3 [cyclo(Asn(1)-Pro(2)-Phe(3)-Ile(4)-Leu(5)-Pro(6)-Val(7))], in DMSO, which were studied by nmr by Mechnich et al. (Helvetica Chimica Acta, 1997, vol. 80, pp. 1338-1354). The calculations for axinastatin 2 show that special ASPs should be optimized for the partially charged side-chain atoms of Asn while the rest of the atoms take their values derived in our previous work; this suggests that similar optimization might be needed for other side chains as well. The solution structures of these peptides are obtained ab initio (i.e., without using experimental restraints) by an extensive conformational search based on E(GRO) alone and E(*)(tot), which consists of the new set of ASPs. For E(*)(tot), the theoretical values of proton-proton distances, (3)J coupling constants, and other properties are found to agree very well with the nmr results, and they are always better than those based on E(GRO).

Ab initio prediction of the solution structures and populations of a cyclic pentapeptide in DMSO based on an implicit solvation model.

Using a recently developed statistical mechanics methodology, the solution structures and populations of the cyclic pentapeptide cyclo(D-Pro(1)-Ala(2)-Ala(3)-Ala(4)-Ala(5)) in DMSO are obtained ab initio, i.e., without using experimental restraints. An important ingredient of this methodology is a novel optimization of implicit solvation parameters, which in our previous publication [Baysal, C.; Meirovitch, H. J Am Chem Soc 1998, 120, 800-812] has been applied to a cyclic hexapeptide in DMSO. The molecule has been described by the simplified energy function E(tot) = E(GRO) + summation operator(k) sigma(k)A(k), where E(GRO) is the GROMOS force-field energy, sigma(k) and A(k) are the atomic solvation parameter (ASP) and the solvent accessible surface area of atom k. This methodology, which relies on an extensive conformational search, Monte Carlo simulations, and free energy calculations, is applied here with E(tot) based on the ASPs derived in our previous work, and for comparison also with E(GRO) alone. For both models, entropy effects are found to be significant. For E(tot), the theoretical values of proton-proton distances and (3)J coupling constants agree very well with the NMR results [Mierke, D. F.; Kurz, M.; Kessler, H. J Am Chem Soc 1994, 116, 1042-1049], while the results for E(GRO) are significantly worse. This suggests that our ASPs might be transferrable to other cyclic peptides in DMSO as well, making our methodology a reliable tool for an ab initio structure prediction; obviously, if necessary, parts of this methodology can also be incorporated in a best-fit analysis where experimental restraints are used.

Impact of Helicobacter pylori infection on serum gastrin in haemodialysis patients.

BACKGROUND: Helicobacter pylori infection is associated with increased gastrin release in patients with normal renal function. Hypergastrinaemia is a common finding in haemodialysis patients and, in many cases, may be linked to H. pylori infection. The aim of this study was to examine the effect of H. pylori infection, and its eradication, on elevated gastrin levels in haemodialysis patients. METHODS: Eighty-nine dyspeptic patients were included in the study. While 44 patients had normal renal function, the remaining 45 were end-stage renal failure patients. Patients were assigned to one of four groups according to their H. pylori and renal function status. Infected patients were re-evaluated after 2 months following eradication treatment. Serum gastrin levels were measured in these groups both before and after eradication treatment. RESULTS: Haemodialysis patients with H. pylori infection had higher serum gastrin levels than did H. pylori negative haemodialysis patients (321+/-131 pg/ml vs 154+/-25 pg/ml) (P<0.05). Mean serum gastrin concentration was 152+/-21 pg/ml in the non-uraemic H. pylori-positive group. This value was 58+/-17 pg/ml in the non-uraemic H. pylori-negative group (P<0.05). There were significant decreases in serum gastrin levels from pre- to post-eradication of H. pylori in the infected haemodialysis and non-uraemic patient groups (312+/-131 pg/ml to 179+/-85 pg/ml and 152+/-21 pg/ml to 72+/-2.4 pg/ml respectively, P<0.05). Four patients in group Ib and 5 patients in group IIb who had persistent infection did not have a decrease in serum gastrin level. All patients with successful eradication had a decrease in serum gastrin concentration. CONCLUSION: Our findings suggest that H. pylori infection contributes to hypergastrinaemia in haemodialysis patients. More research is needed regarding the clinical consequences of hypergastrinaemia in these individuals.

Free energy based populations of interconverting microstates of a cyclic peptide lead to the experimental NMR data.

Analysis of nuclear Overhauser enhancement (NOE) intensities data of interconverting microstates of a peptide is a difficult problem in nmr. A new statistical mechanics methodology has been proposed recently, consisting of several steps: (1) potential energy wells on the energy surface of the molecule are identified (the corresponding regions are called wide microstates); (2) each wide microstate is then spanned by a Monte Carlo (MC) or molecular dynamics simulation starting from a representative structure, and the corresponding relative populations are obtained from the free energy calculated with the local states method; and (3) the overall NOEs and 3J coupling constants are obtained as averages over the corresponding contributions of the samples, weighted by the populations. Extending this methodology to cyclic peptides, we are treating here the hexapeptide cyclo(D-Pro1-Phe2-Ala3-Ser4-Phe5-Phe6) in DMSO, which was studied by Kessler et al. using nmr (Journal of the American Chemical Society, 1992, Vol. 114, pp. 4805-4818). They found that at least two structures are required to explain their NOE data, a conclusion also corroborated by our analysis (Journal of the American Chemical Society, 1998, Vol. 120, pp. 800-812) and led to a novel derivation of atomic solvation parameters (ASPs) for DMSO. Thus, the overall interactions within the peptide-solvent system are described approximately by Etot = EGRO + summation operator sigmaiAi, where EGRO is the energy of the GROMOS force field, Ai is the solvent-accessible surface area of atom i, and sigmai is the ASP. In the present paper the validity of these ASPs within the framework of the entire methodology is verified. This requires taking into account 23 microstates. A very good agreement is obtained between experimental and calculated NOEs and 3J coupling constants. The free energy based populations lead to the best results, which means that entropic effects should not be ignored. We have also studied the behavior of the internal angular fluctuations of the proton-proton vectors and discovered that they have a negligible effect on the calculated NOEs; this is due to the relatively concentrated wide microstates spanned by the MC simulations. The applicability of our ASPs to other cyclic peptides in DMSO is being studied in another work and preliminary results are discussed.