We have compared molecular dynamics (MD) simulations of a β-hairpin forming peptide derived from the protein Nrf2 with 10 biomolecular force fields using trajectories of at least 1 μs. The total simulation time was 37.2 μs. Previous studies have shown that different force fields, water models, simulation methods, and parameters can affect simulation outcomes. The MD simulations were done in explicit solvent with a 16-mer Nrf2 β-hairpin forming peptide using Amber ff99SB-ILDN, Amber ff99SB*-ILDN, Amber ff99SB, Amber ff99SB*, Amber ff03, Amber ff03*, GROMOS96 43a1p, GROMOS96 53a6, CHARMM27, and OPLS-AA/L force fields. The effects of charge-groups, terminal capping, and phosphorylation on the peptide folding were also examined. Despite using identical starting structures and simulation parameters, we observed clear differences among the various force fields and even between replicates using the same force field. Our simulations show that the uncapped peptide folds into a native-like β-hairpin structure at 310 K when Amber ff99SB-ILDN, Amber ff99SB*-ILDN, Amber ff99SB, Amber ff99SB*, Amber ff03, Amber ff03*, GROMOS96 43a1p, or GROMOS96 53a6 were used. The CHARMM27 simulations were able to form native hairpins in some of the elevated temperature simulations, while the OPLS-AA/L simulations did not yield native hairpin structures at any temperatures tested. Simulations that used charge-groups or peptide capping groups were not largely different from their uncapped counterparts with single atom charge-groups. On the other hand, phosphorylation of the threonine residue located at the β-turn significantly affected the hairpin formation. To our knowledge, this is the first study comparing such a large set of force fields with respect to β-hairpin folding. Such a comprehensive comparison will offer useful guidance to others conducting similar types of simulations.

Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and com...

Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.

Prion diseases are disorders that share several characteristics that are typical of many neurodegenerative diseases. Recently, several studies have extended the prion concept to pathological aggregation in malignant tumors involving misfolded p53, a tumor-suppressor protein. The aggregation of p53 and its coaggregation with p53 family members, p63 and p73, have been shown. Certain p53 mutants exert a dominant-negative regulatory effect on wild-type (WT) p53. The basis for this dominant-negative effect is that amyloid-like mutant p53 converts WT p53 into an aggregated species, leading to a gain-of-function (GoF) phenotype and the loss of its tumor-suppressor function. Recently, it was shown that p53 aggregates can be internalized by cells and can coaggregate with endogenous p53, corroborating the prion-like properties of p53 aggregates. The prion-like behavior of oncogenic p53 mutants provides an explanation for its dominant-negative and GoF properties, including the high metastatic potential of cancer cells carrying p53 mutations. The inhibition of p53 aggregation appears to represent a promising target for therapeutic intervention in patients with malignant tumors.