ncIDP-assign: a SPARKY extension for the effective NMR assignment of intrinsically disordered proteins

Summary: We describe here the ncIDP-assign extension for the popular NMR assignment program SPARKY, which aids in the sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The assignment plugin greatly facilitates the effective matching of a set of connected resonances to the correct position in the sequence by making use of IDP random coil chemical shifts

Methods of probing the interactions between small molecules and disordered proteins

It is generally recognized that a large fraction of the human proteome is made up of proteins that remain disordered in their native states. Despite the fact that such proteins play key biological roles and are involved in many major human diseases, they still represent challenging targets for drug discovery. A major bottleneck for the identification of compounds capable of interacting with these proteins and modulating their disease-promoting behaviour is the development of effective techniques to probe such interactions. The difficulties in carrying out binding measurements have resulted in a poor understanding of the mechanisms underlying these interactions. In order to facilitate further methodological advances, here we review the most commonly used techniques to probe three types of interactions involving small molecules: (1) those that disrupt functional interactions between disordered proteins; (2) those that inhibit the aberrant aggregation of disordered proteins, and (3) those that lead to binding disordered proteins in their monomeric states. In discussing these techniques, we also point out directions for future developments.Gabriella T. Heller is supported by the Gates Cambridge Trust Scholarship. Francesco A. Aprile is supported by a Senior Research Fellowship award from the Alzheimer’s Society, UK (grant number 317, AS-SF-16-003)

A method for validating the accuracy of NMR protein structures

We present a method that measures the accuracy of NMR protein structures. It compares random coil index [RCI] against local rigidity predicted by mathematical rigidity theory, calculated from NMR structures [FIRST], using a correlation score (which assesses secondary structure), and an RMSD score (which measures overall rigidity). We test its performance using: structures refined in explicit solvent, which are much better than unrefined structures; decoy structures generated for 89 NMR structures; and conventional predictors of accuracy such as number of restraints per residue, restraint violations, energy of structure, ensemble RMSD, Ramachandran distribution, and clashscore. Restraint violations and RMSD are poor measures of accuracy. Comparisons of NMR to crystal structures show that secondary structure is equally accurate, but crystal structures are typically too rigid in loops, whereas NMR structures are typically too floppy overall. We show that the method is a useful addition to existing measures of accuracy

Probing the free energy landscape of the FBP28 WW domain using multiple techniques

The free-energy landscape of a small protein, the FBP 28 WW domain, has been explored using molecular dynamics (MD) simulations with alternative descriptions of the molecule. The molecular models used range from coarsegrained to all-atom with either an implicit or explicit treatment of the solvent. Sampling of conformation space was performed using both conventional and temperature-replica exchange MD simulations. Experimental chemical shifts and NOEs were used to validate the simulations, and experimental φ values both for validation and as restraints. This combination of different approaches has provided insight into the free energy landscape and barriers encountered by the protein during folding and enabled the characterization of native, denatured and transition states which are compatible with the available experimental data. All the molecular models used stabilize well defined native and denatured basins; however, the degree of agreement with the available experimental data varies. While the most detailed, explicit solvent model predicts the data reasonably accurately, it does not fold despite a simulation time 10 times that of the experimental folding time. The less detailed models performed poorly relative to the explicit solvent model: an implicit solvent model stabilizes a ground state which differs from the experimental native state, and a structure-based model underestimates the size of the barrier between the two states. The use of experimental φ values both as restraints, and to extract structures from unfolding simulations, result in conformations which, although not necessarily true transition states, appear to share the geometrical characteristics of transition state structures. In addition to characterizing the native, transition and denatured states of this particular system in this work, the advantages and limitations of using varying levels of representation are discussed. © 2008 Wiley Periodicals, Inc

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone