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Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.

Proteins that fold rapidly, on the (sub-) microsecond time scale, offer the prospect of direct comparison between experimental data and molecular dynamics simulations. However, experimental studies for such proteins frequently are hindered because folding rates are too fast to measure using conventional stopped-flow methods. To overcome this impediment, NMR spin relaxation dispersion experiments are used to quantify mutational effects on kinetics (DeltaDeltaG(o)), stability (DeltaDeltaG(o)), and phi-values (DeltaDeltaG(dagger)/DeltaDeltaG(o)) for proteins exhibiting chemical exchange line broadening that is fast on the NMR chemical shift time scale. The accuracy of phi-value analysis is enhanced because mutational effects on denatured or intermediate states can be detected through changes in line broadening. The transition and intermediate states of the villin headpiece domain, HP67, are characterized in varying solvent conditions to validate the method.

The traditional view of the denatured state ensemble of proteins is that it behaves as a classic random coil. This model has important implications for the analysis of protein stability, protein folding, and cooperativity; namely that the effects of mutations on the free energy of the denatured state ensemble can be ignored. This assumption, which is still routinely made, at least at the implicit level, greatly simplifies the analysis of such experiments. However it has long been recognized that the denatured state ensemble (DSE) of real proteins is often quite different from a random coil and can exhibit significant structural preferences. In some cases parts of the chain can even adopt relatively well-defined conformations, particularly under native conditions. Well-studied examples of DSE interactions include elements of hydrogen-bonded secondary structure, particularly helices or turns, as well hydrophobic clusters, hydrophobic aromatic clusters, and more recently interactions involving charged residues. Deviations from random-coil behavior are of practical importance if they influence protein folding, stability, or function, or if they compromise our analysis and interpretation of experiments. The existence of residual structure in the DSE naturally leads to the question of its role in protein folding and stability, and raises the possibility that some mutations could exert a significant part of their effect by altering the DSE. Much of our understanding of the interactions governing protein stability and the folding process have been generated by mutational studies; thus, a detailed understanding of the denatured state ensemble is critical.

Analysis of the phi value is one of the most powerful tools to understand the transition state for protein folding. In principle, phi values are expected to fall in the range of 0 to 1. However, a noticeable number of phi values have been observed which are either less than 0 or greater than 1. The origin of such phi values, sometimes referred to as noncanonical or nonclassical phi values, has been controversial. Here we show that mutational effects upon denatured state energetics can lead to nonclassical phi values.

Nonstructural protein 1 (NS1) is a multifunctional virulence factor of influenza virus. The effector domain (ED) of influenza viruses is capable of binding to a variety of host factors, however, the molecular basis of the interactions remains to be investigated. The isolated NS1-ED exists in equilibrium between the monomer and homodimer. Although the structural diversity of the dimer interface has been well-characterized, limited information is available regarding the internal conformational heterogeneity of the monomeric NS1-ED. Here, we present the solution NMR structure of the NS1-ED W187R of the 1918 influenza A virus, which caused the "Spanish flu." Structural plasticity is an essential property to understand the molecular mechanism by which NS1-ED interacts with multiple host proteins. Structural comparison with the NS1-ED from influenza A/Udorn/1972 (Ud) strain revealed a similar overall structure but a distinct conformational variation and flexibility. Our results suggest that conformational flexibility of the NS1-ED might differ depending on the influenza strain. • Solution NMR structure of the NS1-ED of the 1918 influenza A virus. • NS1-ED W187R of the 1918 influenza A virus exists as a monomer in solution. • The 1918 NS1-ED is structurally plastic in the binding sites for host proteins. • Spatial distribution of structurally plastic regions might vary among NS1-ED proteins. [ABSTRACT FROM AUTHOR]

Abstract: The efficient expression of small to midsize polypeptides and small marginally stable proteins can be difficult. A new protein fusion system is developed to allow the expression of peptides and small proteins. The polypeptide of interest is linked via a Factor Xa cleavage sequence to the C-terminus of the N-terminal domain of the ribosomal protein L9 (NTL9). NTL9 is a small (56 residue) basic protein. The C-terminus of the protein is part of an α-helix which extends away from the globular structure thus additional domains can be fused without altering the fold of NTL9. NTL9 expresses at high levels, is extremely soluble, and remains fully folded over a wide temperature and pH range. The protein has a high net positive charge, facilitating purification of fusion proteins by ion exchange chromatography. NTL9 fusions can also be easily purified by reverse phase HPLC. As a test case we demonstrate the high level expression of a small, 36 residue, three helix bundle, the villin headpiece subdomain. This protein is widely used as a model system for folding studies and the development of a simple expression system should facilitate experimental studies of the subdomain. The yield of purified fusion protein is 70mg/L of culture and the yield of purified villin headpiece subdomain is 24mg/L of culture. We also demonstrate the use of the fusion system to express a smaller marginally folded peptide fragment of the villin headpiece domain. [Copyright &y& Elsevier]