Single-molecule Studies Of p53 Sliding Along DNA

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S100B (S100 calcium binding protein B)

Review on S100B (S100 calcium binding protein B), with data on DNA, on the protein encoded, and where the gene is implicated

Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange

AbstractThe extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterised by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 15N-labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen-deuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions

PFD: a database for the investigation of protein folding kinetics and stability

We have developed a new database that collects all protein folding data into a single, easily accessible public resource. The Protein Folding Database (PFD) contains annotated structural, methodological, kinetic and thermodynamic data for more than 50 proteins, from 39 families. A user-friendly web interface has been developed that allows powerful searching, browsing and information retrieval, whilst providing links to other protein databases. The database structure allows visualization of folding data in a useful and novel way, with a long-term aim of facilitating data mining and bioinformatics approaches. PFD can be accessed freely at http://pfd.med.monash.edu.au

High Temperature Unfolding Simulations of the TRPZ1 Peptide

We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 μs with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations. Several unfolding and refolding transition pathways are sampled and analyzed. The unfolding process of the peptide occurs in two steps because of the accumulation of a metastable on-pathway intermediate state stabilized by two native backbone hydrogen bonds assisted by nonnative hydrophobic interactions between the tryptophan side chains. Analysis of the un/folding kinetics and classical commitment probability calculations on the conformations extracted from the transition pathways show that the rate-limiting step for unfolding is the disruption of the ordered native hydrophobic packing (Trp-zip motif) leading from the native to the intermediate state. But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues. The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments. The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here

Exploiting transient protein states for the design of small-molecule stabilizers of mutant p53

The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-α-helical bundle. The holo protein apparently refolds with a half-life of 4 μs in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 μs that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.This work was supported by the BBSRC under the SBDA initiative, the EU TMR Life Sciences programme (contract ERBFMRX-CT-98-0230), the MRC and the NIH (grant GM056250 to H.R.). P.D.B. thanks the BBSRC for an Advanced Research Fellowshi

Targeting cavity-creating p53 cancer mutations with small-molecule stabilizers: the Y220X paradigm

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket’s accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutant as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therap

Small molecule induced reactivation of mutant p53 in cancer cells

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Here, we have identified a compound, PK7088, which is active in vitro: PK7088 bound to the mutant with a dissociation constant of 140 μM and raised its melting temperature, and we have determined the binding mode of a close structural analogue by X-ray crystallography. We showed that PK7088 is biologically active in cancer cells carrying the Y220C mutant by a battery of tests. PK7088 increased the amount of folded mutant protein with wild-type conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. We suggest a set of criteria for assigning activation of p53