Prion domain initiation of amyloid formation in vitro from native Ure2p.
The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diameter with more than 60 percent beta sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in extracts of [URE3] strains, these 180- to 220-angstrom-diameter filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymerization of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3]. (+info)
Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions.
Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues. (+info)
Low temperature and pressure stability of picornaviruses: implications for virus uncoating.
The family Picornaviridae includes several viruses of great economic and medical importance. Poliovirus replicates in the human digestive tract, causing disease that may range in severity from a mild infection to a fatal paralysis. The human rhinovirus is the most important etiologic agent of the common cold in adults and children. Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases in cattle. These viruses have in common a capsid structure composed of 60 copies of four different proteins, VP1 to VP4, and their 3D structures show similar general features. In this study we describe the differences in stability against high pressure and cold denaturation of these viruses. Both poliovirus and rhinovirus are stable to high pressure at room temperature, because pressures up to 2.4 kbar are not enough to promote viral disassembly and inactivation. Within the same pressure range, FMDV particles are dramatically affected by pressure, with a loss of infectivity of more than 4 log units observed. The dissociation of polio and rhino viruses can be observed only under pressure (2.4 kbar) at low temperatures in the presence of subdenaturing concentrations of urea (1-2 M). The pressure and low temperature data reveal clear differences in stability among the three picornaviruses, FMDV being the most sensitive, polio being the most resistant, and rhino having intermediate stability. Whereas rhino and poliovirus differ little in stability (less than 10 kcal/mol at 0 degrees C), the difference in free energy between these two viruses and FMDV was remarkable (more than 200 kcal/mol of particle). These differences are crucial to understanding the different factors that control the assembly and disassembly of the virus particles during their life cycle. The inactivation of these viruses by pressure (combined or not with low temperature) has potential as a method for producing vaccines. (+info)
Tolerance of Arc repressor to multiple-alanine substitutions.
Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure. (+info)
Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate.
On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate. (+info)
A specific transition state for S-peptide combining with folded S-protein and then refolding.
We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the alpha-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the alpha-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of -0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state. (+info)
Stretching lattice models of protein folding.
A new class of experiments that probe folding of individual protein domains uses mechanical stretching to cause the transition. We show how stretching forces can be incorporated in lattice models of folding. For fast folding proteins, the analysis suggests a complex relation between the force dependence and the reaction coordinate for folding. (+info)
Analysis of protein-protein interactions by mutagenesis: direct versus indirect effects.
Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information. (+info)