Native molten globules are the most folded kind of intrinsically disordered proteins. Little is known about the mechanism by which native molten globules bind to their cognate ligands to form fully folded complexes. The nuclear coactivator binding domain (NCBD) of CREB binding protein is particularly interesting in this respect as structural studies of its complexes have shown that NCBD folds into two remarkably different states depending on the ligand being ACTR or IRF-3. The ligand-free state of NCBD was characterized in order to understand the mechanism of folding upon ligand binding. Biophysical studies show that despite the molten globule nature of the domain, it contains a small cooperatively folded core. By NMR spectroscopy, we have demonstrated that the folded core of NCBD has a well ordered conformer with specific side chain packing. This conformer resembles the structure of the NCBD in complex with the protein ligand, ACTR, suggesting that ACTR binds to prefolded NCBD molecules from the ensemble of interconverting structures.CREB binding protein | folding upon binding | NMR spectroscopy T he protein structure-function paradigm has dominated structural and molecular biology over the past five decades. This paradigm has been challenged by the discovery of functional but intrinsically disordered proteins (IDPs) (1). IDPs are abundant in higher organisms and are necessary for many crucial biological functions such as cell cycle regulation, signal transduction, and regulation of transcription (2, 3). Structural studies of IDPs have shown that despite the high degree of disorder, these proteins are far from randomly structured and form transient, yet specific, secondary and tertiary structural elements (4, 5). The success of the structure-function paradigm in explaining the function of folded proteins suggests that the functions of the IDPs may also be understood by studying the transient structure of the disordered state.The molten globule was originally discovered as a partially folded state under mildly denaturing conditions and was shown to accumulate as an intermediate during protein folding reactions (6, 7). With the emergence of the IDPs, proteins were discovered where the molten globule is the functionally active state. Native molten globules are the most compact conformational state considered as IDPs (4). Molten globules have native-like secondary structure, but are believed to lack the well-defined tertiary interactions found in folded proteins. Most high resolution NMR studies of the molten globule state have focused on the secondary structure of the backbone as this is more readily accessible from secondary chemical shifts (8). The degree of side chain packing, however, is less well understood. Molten globules are believed to have dynamic hydrophobic cores due to the lack of signal in the near-UV CD spectrum and the broadening of NMR signals of aromatic groups (6, 7). Recent studies have shown that the side chains in the α-lactalbumin molten globule exchange between several well defined confor...

Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins. The quality of the secondary chemical shifts is dependent on an appropriate choice of random coil chemical shifts. We report random coil chemical shifts and sequence correction factors determined for a GGXGG peptide series following the approach of Schwarzinger et al. (J Am Chem Soc 123(13):2970-2978, 2001). The chemical shifts are determined at neutral pH in order to match the conditions of most studies of intrinsically disordered proteins. Temperature has a non-negligible effect on the (13)C random coil chemical shifts, so temperature coefficients are reported for the random coil chemical shifts to allow extrapolation to other temperatures. The pH dependence of the histidine random coil chemical shifts is investigated in a titration series, which allows the accurate random coil chemical shifts to be obtained at any pH. By correcting the random coil chemical shifts for the effects of temperature and pH, systematic biases of the secondary chemical shifts are minimized, which will improve the reliability of detection of transient secondary structure in disordered proteins.

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient a-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.Keywords: intrinsically disordered protein (IDP); transient secondary structure; temperature dependence; polyproline II; circular dichroism (CD); nuclear magnetic resonance spectroscopy (NMR); sodium-proton (Na1/H1) exchanger 1 (NHE1); S-phase delayed 1 (Spd1); activator for thyroid hormone and retinoid receptors (ACTR) Abbreviations: ACTR, activator for thyroid hormone and retinoid receptors; CBP, CREB-binding protein; CD, circular dichroism; IDP, intrinsically disordered protein; NCBD, nuclear coactivator binding domain; NHE1cdt, sodium-proton (Na þ /H þ ) exchanger 1 C-terminal distal tail; NMR, nuclear magnetic resonance; PPII, polyproline II; RDC, residual dipolar coupling; SAXS, small-angle X-ray scattering; Spd1, S-phase delayed 1.

Random coil chemical shifts are necessary for secondary chemical shift analysis, which is the main NMR method for identification of secondary structure in proteins. One of the largest challenges in the determination of random coil chemical shifts is accounting for the effect of neighboring residues. The contributions from the neighboring residues are typically removed by using neighbor correction factors determined based on each residue's effect on glycine chemical shifts. Due to its unusual conformational freedom, glycine may be particularly unrepresentative for the remaining residue types. In this study, we use random coil peptides containing glutamine instead of glycine to determine the random coil chemical shifts and the neighbor correction factors. The resulting correction factors correlate to changes in the populations of the major wells in the Ramachandran plot, which demonstrates that changes in the conformational ensemble are an important source of neighbor effects in disordered proteins. Glutamine derived random coil chemical shifts and correction factors modestly improve our ability to predict (13)C chemical shifts of intrinsically disordered proteins compared to existing datasets, and may thus improve the identification of small populations of transient structure in disordered proteins.

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.