PubMed · 2447649
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D M Barnes. 1988-01-15. NMDA receptors trigger excitement.. https://doi.org/10.1126/science.2447649
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G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific beta-hairpin within 6 micros. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a multicanonical replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like beta-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high phi-values of the turn region observed by experiment.
Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXI(P161L). k(cat) for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is approximately 3-fold lower for activated FXI(P161L) (FXIa(P161L)) than for wild-type FXIa (FXIa(WT)), consistent with an abnormal protease active site. Inhibition of FXIa(P161L) by diisopropyl fluorophosphate is 2.4-fold slower than for FXIa(WT), suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIa(WT) and FXIa(P161L). Rates of carbamylation of Ile16 were also similar for FXIa(WT) and FXIa(P161L), indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.
Five residues in the multifunctional catalase-peroxidase KatG of Burkholderia pesudomallei are essential for catalase, but not peroxidase, activity. Asp141 is the only one of these catalase-specific residues not related with the covalent adduct found in KatGs that when replaced with a nonacidic residue reduces catalase activity to 5% of native levels. Replacing the nearby catalytic residue Arg108 causes a reduction in catalase activity to 35% of native levels, whereas a variant with both Asp141 and Arg108 replaced exhibits near normal catalase activity (82% of native), suggesting a synergism in the roles of the two residues in support of catalase activity in the enzyme. Among the Asp141 variants, D141E is unique in retaining normal catalase activity but with modified kinetics, suggesting more favorable compound I formation and less favorable compound I reduction. The crystal structure of the D141E variant has been determined at 1.8-A resolution, revealing that the carboxylate of Glu141 is moved only slightly compared with Asp141, but retains its hydrogen bond interaction with the main chain nitrogen of Ile237. In contrast, the low temperature ferric Electron Paramagnetic Resonance spectra of the D141A, R108A, and R108A/D141A variants are consistent with modifications of the water matrix and/or the relative positioning of the distal residue side chains. Such changes explain the reduction in catalase activity in all but the double variant R108A/D141A. Two pathways of hydrogen bonded solvent lead from the entrance channel into the heme active site, one running between Asp141 and Arg108 and the second between Asp141 and the main chain atoms of residues 237-239. It is proposed that binding of substrate H(2)O(2) to Asp141 and Arg108 controls H(2)O(2) access to the heme active site, thereby modulating the catalase reaction.