Limited internal friction in the rate-limiting step of a two-state protein folding reaction

Small, single-domain proteins typically fold via a compact transition-state ensemble in a process well fitted by a simple, two-state model. To characterize the rate-limiting conformational changes that underlie two-state folding, we have investigated experimentally the effects of changing solvent viscosity on the refolding of the IgG binding domain of protein L. In conjunction with numerical simulations, our results indicate that the rate-limiting conformational changes of the folding of this domain are strongly coupled to solvent viscosity and lack any significant “internal friction” arising from intrachain collisions. When compared with the previously determined solvent viscosity dependencies of other, more restricted conformational changes, our results suggest that the rate-limiting folding transition involves conformational fluctuations that displace considerable amounts of solvent. Reconciling evidence that the folding transition state ensemble is comprised of highly collapsed species with these and similar, previously reported results should provide a significant constraint for theoretical models of the folding process.

Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion

We have reported some type II restriction-modification (RM) gene complexes on plasmids resist displacement by an incompatible plasmid through postsegregational host killing. Such selfish behavior may have contributed to the spread and maintenance of RM systems. Here we analyze the role of regulatory genes (C), often found linked to RM gene complexes, in their interaction with the host and the other RM gene complexes. We identified the C gene of EcoRV as a positive regulator of restriction. A C mutation eliminated postsegregational killing by EcoRV. The C system has been proposed to allow establishment of RM systems in new hosts by delaying the appearance of restriction activity. Consistent with this proposal, bacteria preexpressing ecoRVC were transformed at a reduced efficiency by plasmids carrying the EcoRV RM gene complex. Cells carrying the BamHI RM gene complex were transformed at a reduced efficiency by a plasmid carrying a PvuII RM gene complex, which shares the same C specificity. The reduction most likely was caused by chromosome cleavage at unmodified PvuII sites by prematurely expressed PvuII restriction enzyme. Therefore, association of the C genes of the same specificity with RM gene complexes of different sequence specificities can confer on a resident RM gene complex the capacity to abort establishment of a second, incoming RM gene complex. This phenomenon, termed “apoptotic mutual exclusion,” is reminiscent of suicidal defense against virus infection programmed by other selfish elements. pvuIIC and bamHIC genes define one incompatibility group of exclusion whereas ecoRVC gene defines another.

Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45

Apoptotic DNA fragmentation is mediated by a caspase-activated DNA fragmentation factor (DFF)40. Expression and folding of DFF40 require the presence of DFF45, which also acts as a nuclease inhibitor before DFF40 activation by execution caspases. The N-terminal domains (NTDs) of both proteins are homologous, and their interaction plays a key role in the proper functioning of this two-component system. Here we report that the NTD of DFF45 alone is unstructured in solution, and its folding is induced upon binding to DFF40 NTD. Therefore, folding of both proteins regulates the formation of the DFF40/DFF45 complex. The solution structure of the heterodimeric complex between NTDs of DFF40 and DFF45 reported here shows that the mutual chaperoning includes the formation of an extensive network of intermolecular interactions that bury a hydrophobic cluster inside the interface, surrounded by intermolecular salt bridges.

Rock friction and its implications for earthquake prediction examined via models of Parkfield earthquakes.

The friction of rocks in the laboratory is a function of time, velocity of sliding, and displacement. Although the processes responsible for these dependencies are unknown, constitutive equations have been developed that do a reasonable job of describing the laboratory behavior. These constitutive laws have been used to create a model of earthquakes at Parkfield, CA, by using boundary conditions appropriate for the section of the fault that slips in magnitude 6 earthquakes every 20-30 years. The behavior of this model prior to the earthquakes is investigated to determine whether or not the model earthquakes could be predicted in the real world by using realistic instruments and instrument locations. Premonitory slip does occur in the model, but it is relatively restricted in time and space and detecting it from the surface may be difficult. The magnitude of the strain rate at the earth's surface due to this accelerating slip seems lower than the detectability limit of instruments in the presence of earth noise. Although not specifically modeled, microseismicity related to the accelerating creep and to creep events in the model should be detectable. In fact the logarithm of the moment rate on the hypocentral cell of the fault due to slip increases linearly with minus the logarithm of the time to the earthquake. This could conceivably be used to determine when the earthquake was going to occur. An unresolved question is whether this pattern of accelerating slip could be recognized from the microseismicity, given the discrete nature of seismic events. Nevertheless, the model results suggest that the most likely solution to earthquake prediction is to look for a pattern of acceleration in microseismicity and thereby identify the microearthquakes as foreshocks.

Dynamic friction and the origin of the complexity of earthquake sources.

We study a simple antiplane fault of finite length embedded in a homogeneous isotropic elastic solid to understand the origin of seismic source heterogeneity in the presence of nonlinear rate- and state-dependent friction. All the mechanical properties of the medium and friction are assumed homogeneous. Friction includes a characteristic length that is longer than the grid size so that our models have a well-defined continuum limit. Starting from a heterogeneous initial stress distribution, we apply a slowly increasing uniform stress load far from the fault and we simulate the seismicity for a few 1000 events. The style of seismicity produced by this model is determined by a control parameter associated with the degree of rate dependence of friction. For classical friction models with rate-independent friction, no complexity appears and seismicity is perfectly periodic. For weakly rate-dependent friction, large ruptures are still periodic, but small seismicity becomes increasingly nonstationary. When friction is highly rate-dependent, seismicity becomes nonperiodic and ruptures of all sizes occur inside the fault. Highly rate-dependent friction destabilizes the healing process producing premature healing of slip and partial stress drop. Partial stress drop produces large variations in the state of stress that in turn produce earthquakes of different sizes. Similar results have been found by other authors using the Burridge and Knopoff model. We conjecture that all models in which static stress drop is only a fraction of the dynamic stress drop produce stress heterogeneity.