An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils.

At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, which results in the formation of insoluble fibrils. 1H-15N HSQC, 3D HSQC-NOESY, and 15N T2 NMR experiments have been used to characterize the soluble components of this reaction. The kinetics of self-assembly show a lag phase followed by an exponential increase in polymerization. A single set of 1H-15N HSQC cross-peaks, corresponding to acid-denatured monomers, is observed during the entire course of the reaction. Under lag phase conditions, 15N resonances of residues that constitute the beta-strands of native CspA are selectively broadened with increasing protein concentration. The dependence of 15N T2 values on spin echo period duration demonstrates that line broadening is due to fast NMR exchange between acid-denatured monomers and soluble aggregates. Exchange contributions to T2 relaxation correlate with the squares of the chemical shift differences between native and acid-denatured CspA, and point to a stabilization of native-like structure upon aggregation. Time-dependent changes in 15N T2 relaxation accompanying the exponential phase of polymerization suggest that the first three beta-strands may be predominantly responsible for association interfaces that promote aggregate growth. CspA serves as a useful model system for exploring the conformational determinants of denatured protein misassembly.

Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide.

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO). The structure of CspA has five strands of beta-sheet. Strands beta1-beta4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all-or-none global unfolding mechanism. By contrast, the protection of amide protons from strand beta5 is too weak to measure in water. Strand beta5 is hydrogen bonded to strands beta3 and beta4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands beta1-beta4 but fail to account for the weak protection in strand beta5. The most conspicuous feature of strand beta5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands beta1-beta4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand beta5 and the loops connecting the beta-strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.

A Plasmodium falciparum protein kinase with two unusually large kinase inserts.

A protein kinase gene (PfPK1) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The gene, which contains one intron, encodes a protein with a predicted length of 909 amino acids. The predicted protein contains all the conserved sequences characteristic of a protein kinase catalytic domain. These sequences are discontinuous, however, since they are separated by two large kinase inserts with 178 and 330 amino acids in size. Specific antisera were raised against recombinant fragments of the protein and a PfPK1-specific peptide. Using one of these antibodies, a functional protein kinase was precipitated from malarial lysates and this kinase recognized casein as an exogenous substrate. PfPK1 was expressed in a stage-specific fashion and also had a stage-specific cellular localization. During the intraerythrocytic life cycle, PfPK1 shifts from the parasite cytosol to the parasite membrane fraction. An unusual feature of PfPK1 is its electrophoretic mobility on SDS-PAGE. Whereas the predicted protein size is about 100 kDa, the apparent size is about 70 kDa. There are no indications for RNA processing and we could exclude proteolytic processing as an explanation.