Effect of protein disulfide isomerase on the rate-determining steps of the folding of bovine pancreatic ribonuclease A

To address the effect of an agglutogen on virus infection, we studied the avidin-associated inhibition of infection by biotinylated M13 phages (BIO-phages). Microscopic observation of mixtures of BIO-phages and avidin–fluorescein conjugates revealed many aggregates. Even at low phage concentrations, avidin induced inhibition of infection significantly. Anti-M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin. The inhibition by avidin was at ≥2 μg/ml, time dependent and marked until 10 min after the mixing of the BIO-phages and Escherichia coli. On the other hand, antibody inhibited the infection at ≥0.1 μg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations. These results indicate that avidin against BIO-phages and antibodies are agglutogens, and the inhibition of the BIO-phages by avidin is closely related to the tetramerization of avidin. Agglutogens may be novel alternative antiviral drugs.     

Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

Exact solutions for chemical bond orientations from residual dipolar couplings

New methods for determining chemical structures from residual dipolar couplings are presented. The fundamental dipolar coupling equation is converted to an elliptical equation in the principal alignment frame. This elliptical equation is then combined with other angular or dipolar coupling constraints to form simple polynomial equations that define discrete solutions for the unit vector(s). The methods are illustrated with residual dipolar coupling data on ubiquitin taken in a single anisotropic medium. The protein backbone is divided into its rigid groups (namely, its peptide planes and C frames), which may be solved for independently. A simple procedure for recombining these independent solutions results in backbone dihedral angles and that resemble those of the known native structure. Subsequent refinement of these - angles by the ROSETTA program produces a structure of ubiquitin that agrees with the known native structure to 1.1 Å C rmsd.     

Formation of the hydrophobic core of ribonuclease A through sequential coordinated conformational transitions

With steady-state and time-resolved fluorescence energy-transfer measurements, we determined the distributions of intramolecular distances in nine mutants to study the conformations of wild-type ribonuclease A in the reduced state under folding conditions. Although far-UV-CD measurements show no evidence for a secondary-structure transition, temperature- and GdnHCl-induced changes in intramolecular distance distributions in the reduced state revealed evidence for long-range subdomain structures in the denatured protein. These poorly defined structures, reflected here by wide distributions corresponding to a wide range of energies, form during refolding in a complex sequence of multiple subdomain transitions. A more well-defined structure emerges only when this structural framework, which directs the successive steps in the folding process, matures and is reinforced by stronger interactions such as disulfide bonds.     

Molecular simulation study of cooperativity in hydrophobic association: clusters of four hydrophobic particles

The multibody contribution to the potential of mean force (PMF) of hydrophobic association of four methane molecules in water was investigated by means of umbrella-sampling molecular dynamics. Two systems were considered: (i). a trigonal pyramid with three methane molecules at contact distance forming a fixed base, the fourth molecule being placed on the top with variable distance from the base; and (ii). a regular uniformly expanding tetrahedron. Methane-methane distances as far as 12.5 A, i.e. beyond the second solvent-separated minimum of the PMF, were considered to address the baseline problem. In contrast to the small effect in the three-body case studied previously (Protein Sci 9 (2000) 1235), the multibody contribution was found to amount to approximately 0.2 kcal/mol per methane-methane pair, or approximately 25% of the depth of the contact minimum in the PMF. The main effect of the multibody contribution to the PMF is a reduction of the height of the barrier between the contact and solvent separated minima and a narrowing of the region of its maximum, while the region of the contact minimum is affected only weakly. The reduction of the barrier is due to four-body contributions. The cooperative contributions to the PMF agree very well with those computed from the molecular surface of the systems under consideration, which further supports earlier observations that the molecular surface can be used with good accuracy to describe the energetics of hydrophobic association.     

Unblocked statistical-coil tetrapeptides in aqueous solution: quantum-chemical computation of the carbon-13 NMR chemical shifts

We recently reported a theoretical characterization of representative ensembles of statistical-coil conformations for tetrapeptides with unblocked termini in aqueous solution, at pH 7. The results showed good agreement between the computed Boltzmann-averaged and experimentally-determined values for both the vicinal coupling constants ³J_NH and the -proton chemical shifts. Here, we carry out a cluster analysis of the ensembles of conformations generated in that study, and use them to compute the Boltzmann-averaged values of the quantum-chemical ¹³C chemical shifts for different amino acids in the unblocked tetrapeptides GGXA (where X stands for Phe, Arg, His, Glu, Ile, Lys, Gln, Tyr, Leu, Thr, Ala, Gly and Val). The values of the ¹³C chemical shifts in these thirteen amino acids (for which experimental data are available) were computed by using Density Functional Theory with a 6–311+G(2d,p) basis set. Good agreement is found in terms of both the correlation coefficient (R) and standard deviations of the difference between the computed Bolztmann-averaged and the NMR-determined values for the ¹³C chemical shifts. These results suggest that it may be possible to build a reliable theoretically-derived database of chemical shifts for statistical-coil residues. The results of the current study contribute to our understanding of the relations between chemical shifts, dihedral angles and vicinal coupling constants, ³J_NH. In addition, they can shed light as to how the statistical-coilconformation is related to the conformational preference of more structured states, such as the -helical conformation.     

Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp96. Implications of the Hydrophobic Interaction Between Trp96 and Pro60B Pro60C for the Activity of Thrombin

A number of thrombin mutants have been constructed to investigate the role of Trp⁹⁶ and the β-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the β-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the β-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp⁹⁶→Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than thrombin, with the two β-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 μM^?1 s^?1 respectively, compared to 10 and 2.5 μM^?1 s^?1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant K _m increased ~6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the β-insertion loop is important for thrombin activity. But the mutation of Trp⁹⁶→Ser can compensate somewhat for the loss of binding at the β-insertion loop. The deletion of the hydrophobic interaction between Trp⁹⁶ and Pro^60BPro^60C appears to decrease the stability of the β-insertion loop, thereby causing a decrease in binding efficiency.     

Implementation of a k/k(0) method to identify long-range structure in transition states during conformational folding/unfolding of proteins

A previously introduced kinetic-rate constant (k/k(0)) method, where k and k(0) are the folding (unfolding) rate constants in the mutant and the wild-type forms, respectively, of a protein, has been applied to obtain qualitative information about structure in the transition state ensemble (TSE) of bovine pancreatic ribonuclease A (RNase A), which contains four native disulfide bonds. The method compares the folding (unfolding) kinetics of RNase A, with and without a covalent crosslink and tests whether the crosslinked residues are associated in the folding (unfolding) transition state (TS) of the noncrosslinked version. To confirm that the fifth disulfide bond has not introduced a significant structural perturbation, we solved the crystal structure of the V43C-R85C mutant to 1.6 A resolution. Our findings suggest that residues Val43 and Arg85 are not associated, and that residues Ala4 and Val118 may form nonnative contacts, in the folding (unfolding) TSE of RNase A.     

Common functionally important motions of the nucleotide‐binding domain of Hsp70

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate‐binding domain (SBD) that binds client substrates, and the nucleotide‐binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure–function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all‐atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP‐ and ATP‐unique classes, which reflect conformational trends that are unique to either the ADP‐ or ATP‐bound states, respectively. “Mutual” class motions generally describe “in‐plane” and/or “out‐of‐plane” (scissor‐like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The “unique” class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the “unique” type, regions of enhanced mobility can be identified; these are termed “hot spots,” and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide‐binding pocket was also found to influence the dynamics of the NBD significantly. Proteins 2015; 83:282–299. © 2014 Wiley Periodicals, Inc.