No abstract

We report the combined use of steady-state fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to investigate conformational distributions of the prion protein (PrP) repeat system. FRET was used for the first time to probe the distance, as a function of temperature and pH, between a donor Trp residue and an acceptor dansyl group attached to the N-terminus in seven model peptides containing one to three repeats of the second decarepeat of PrP from marsupial possum (PHPGGSNWGQ)nG, and one and two human PrP consensus octarepeats (PHGGGWGQ)nG. In multirepeat peptides, single-Trp mutants were made by replacing other Trp(s) with Phe. As previous work has shown PrP repeats do not adopt a single preferred stable conformation, the FRET values are averages reflecting heterogeneity in the donor-acceptor distances. The T-dependence of the conformational distributions, and derived average dansyl-Trp distances, were obtained directly from MD simulation of the marsupial dansyl-PHPGGSNWGQG peptide. The results show excellent agreement between the FRET and MD T-dependent distances, and demonstrate the remarkable sensitivity and reproducibility of the FRET method in this first-time use for a set of disordered peptides. Based on the results, we propose a model involving cation-pi or pi-pi His-Trp interactions to explain the T- (5-85 degrees C) and pH- (6.0, 7.2) dependencies on distance, with HW i, i + 4 or WH i, i + 4 separations in sequence being more stable than HW i, i + 6 or WH i, i + 6 separations. The model has peptides adopting loosely folded conformations, with dansyl-Trp distances very much less than estimates for fully extended conformations, for example, approximately 16 vs. 33, approximately 21 vs. 69, and approximately 22 vs. 106 A for 1-3 decarepeats, and approximately 14 vs. 25 and approximately 19 vs. 54 A for 1-2 octarepeats, respectively. The study demonstrates the usefulness of combining FRET with MD, a combination reported only once previously. Initial "mapping" of the conformational distribution of flexible peptides by simulation can assist in designing and interpreting experiments using steady-state intensity methods, and indicating how time-resolved or anisotropy methods might be used.

The highly conserved nature and tissue specificity of the seven vertebrate beta-tubulin isotypes provide circumstantial evidence that functional differences among isotypes may exist in vivo. Compelling evidence from studies of bovine brain beta-isotypes indicated significant conformational and functional differences in vitro and implied that these differences could be related to in vivo function. A previously uninvestigated parameter of potential importance in assessing functional significance is molecular stability. We examined the relative stability of alphabetaII and alphabetaIII tubulin dimers purified from bovine brain. The use of probes to monitor the exposure of hydrophobic areas and sulfhydryls and the loss of colchicine binding, all of which are known to accompany tubulin's time-dependent loss of function, showed an acceleration of these criteria in alphabetaII relative to alphabetaIII when the isotypes were incubated at 37 degrees C. Studies using differential scanning calorimetry suggested that unfolding of the isotypes at approximately 60 degrees C and decay at 0 degrees C were both highly cooperative. It was also observed that alphabetaIII had a higher melting temperature and a larger population of molecules retaining tertiary structure after incubation at 0 degrees C for 20 h. These studies support the conclusion that alphabetaIII is significantly more stable than alphabetaII and raise the possibility that differences in relative stabilities of tubulin isotypes may be important in regulating the functional properties of microtubules in vivo.

The effects of regional sequence differences on the thermodynamic stability of a globular protein have been investigated by scanning microcalorimetry. Thermal transitions have been measured for two isozymes of yeast cytochrome c (iso-1-MS and iso-2) and three composite proteins (Comp1-MS, Comp2-MS, and Comp3-MS) in which amino acid segments are exchanged between the parental isozymes. There are three main observations. (1) In the temperature range of the unfolding transitions (40-60 degrees C) the unfolding free energies for the composite proteins are only slightly different from those of the parental isozymes, although in some cases there are large but compensating changes in the transitional enthalpy and entropy. At lower temperatures (0-30 degrees C), all the composites are significantly less stable than the two parental proteins. (2) Long-range structural effects are responsible for at least some of the observed differences in stability. For example, in the temperature range of the unfolding transitions (40-60 degrees C), the Comp1-MS protein which contains only a small amount of iso-2-like sequence is less stable than either of the parental isozymes, despite the fact that none of the iso-2-specific amino acid side chains impinges directly on any of the iso-1-specific amino acid side chains. (3) Changes in ionization of His 26 appear to be linked to thermal unfolding. Iso-1-MS and Comp1-MS contain a histidine residue at position 26 while iso-2 and the other two composites do not. On lowering the pH from pH 6 to 5, both iso-1-MS and Comp1-MS show a decrease in stability (lower Tm) within the unfolding transition region (40-60 degrees C), whereas the stabilities of iso-2, Comp2-MS, and Comp3-MS are essentially unchanged. The thermal unfolding transitions are highly reversible (> 95%) but mechanistically complex. A moderate dependence of Tm on protein concentration and the ratio of the van't Hoff enthalpy to the calorimetric enthalpy suggest that thermal unfolding involves the reversible association of a significant fraction of the unfolded species, at least at elevated protein concentrations.

The relationship between structure and stability has been investigated for the folded forms and the unfolded forms of iso-2 cytochrome c and a variant protein with a stability-enhancing mutation, N52I iso-2. Differential scanning calorimetry has been used to measure the reversible unfolding transitions for the proteins in both heme oxidation states. Reduction potentials have been measured as a function of temperature for the folded forms of the proteins. The combination of measurements of thermal stability and reduction potential gives three sides of a thermodynamic cycle and allows prediction of the reduction potential of the thermally unfolded state. The free energies of electron binding for the thermally unfolded proteins differ from those expected for a fully unfolded protein, suggesting that residual structure modulates the reduction potential. At temperatures near 50 degrees C the N52I mutation has a small but significant effect on oxidation state-sensitive structure in the thermally unfolded protein. Inspection of the high-resolution X-ray crystallographic structures of iso-2 and N52I iso-2 shows that the effects of the N52I mutation and oxidation state on native protein stability are correlated with changes in the mobility of specific polypeptide chain segments and with altered hydrogen bonding involving a conserved water molecule. However, there is no clear explanation of oxidation state or mutation-induced differences in stability of the proteins in terms of observed changes in structure and mobility of the folded forms of the proteins alone.