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The traditional structure-function paradigm has provided significant insights for well-folded proteins in which structures can be easily and rapidly revealed by X-ray crystallography beamlines. However, approximately one-third of the human proteome is comprised of intrinsically disordered proteins and regions (IDPs/IDRs) that do not adopt a dominant well-folded structure, and therefore remain "unseen" by traditional structural biology methods. This Perspective considers the challenges raised by the "Dark Proteome", in which determining the diverse conformational substates of IDPs in their free states, in encounter complexes of bound states, and in complexes retaining significant disorder requires an unprecedented level of integration of multiple and complementary solution-based experiments that are analyzed with state-of-the art molecular simulation, Bayesian probabilistic models, and high-throughput computation. We envision how these diverse experimental and computational tools can work together through formation of a "computational beamline" that will allow key functional features to be identified in IDP structural ensembles.

The glycosylation of threonine of the repeating unit of RNA polymerase II with beta-linked N-acetylglucosame was investigated. Two analogues of the repeating C-terminal domain of RNA polymerase II were synthesized and their conformations in water were examined. Experimental results showed that glycosylation of the local, randomly coiled peptide with one biologically essential sugar results in the formation of a turnlike structure. The experiment was the first structural investigation of a novel class of glycoproteins monoglycosylated with N-acetylglucosamine on threonine.

An ensemble approach to the boundary element method that accurately predicts rotational tumbling time ([tau]c) for well-structured and rigid proteins by introducing two layers of molecular surfaces whose correlated velocities decay exponentially with distance is presented. The reliable prediction obtained by ([tau]c would help to detect intra- and intermolecular interactions and conformational switches between more ordered and less ordered states of the disordered segments.

Different methods are used for measuring relaxation dispersion in cases of very tight binding or loss of resonances in the bound state owing to intermediate exchange. The relaxation dispersion measurements for free ligand resonances have provided chemical shifts and structural information for the protein complex.

Examination of a linear peptide with a type VI turn reveals the presence of sterical hindrance for the interactions of few of the proline ring protons with water solvent. NMR experiments suggest that close, specific packing between the proline and aromatic rings in the folded type VI turn conformation is responsible for the reduced accessibility of water to the proline ring protons. This differential hydration confirms that hydrophobic interactions between the aromatic and proline rings stabilize the turn structure.

Pneumatically-assisted electrospray mass spectrometry helps study the binding in the complex between the catalytic antibody NPN43C9 as a cloned single-chain F(v), and its hapten. Analysis at 70 V reveals the tight association between hapten and F(v) and analysis at 175 V reveals noncovalent interactions in the complex. Analysis at high declustering potentials reveals the differences between the hapten-antibody and inhibitor-antibody complex and the results suggests site-specific hapten binding.

For well-structured, rigid proteins, the prediction of rotational tumbling time (vs) using atomic coordinates is reasonably accurate, but is inaccurate for proteins with long unstructured sequences. Under physiological conditions, many proteins contain long disordered segments that play important regulatory roles in fundamental biological events including signal transduction and molecular recognition. Here we describe an ensemble approach to the boundary element method that accurately predicts τc for such proteins by introducing two layers of molecular surfaces whose correlated velocities decay exponentially with distance. Reliable prediction of τc will help to detect intra- and intermolecular interactions and conformational switches between more ordered and less ordered states of the disordered segments. The method has been extensively validated using 12 reference proteins with 14 to 103 disordered residues at the N- and/or C-terminus and has been successfully employed to explain a set of published results on a system that incorporates a conformational switch. [ABSTRACT FROM AUTHOR]