Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.

Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp62 in nonnative and long-range interactions.

Defect-rich ultrathin ZnAl-layered double hydroxide nanosheets are successfully prepared. Under UV-vis irradiation, these nanosheets are superior efficient catalysts for the photoreduction of CO2 to CO with water. The formed oxygen vacancies lead to the formation of coordinatively unsaturated Zn(+) centers within the nanosheets, responsible for the very high photocatalytic activities.

Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although not rationalized. Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation. Importantly, using ion mobility-mass spectrometry (IM-MS), we find that all four macromolecular complexes retain their native-like topologies at low energy. Upon increasing the collision energy, two of the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally. To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits. Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP. The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging. This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.

Oxidized and reduced hen lysozyme denatured in 8 M urea at low pH have been studied in detail by NMR methods. 15N correlated NOESY and TOCSY experiments have provided near complete sequential assignment for both 1H and 15N resonances. Over 900 NOEs, including 130 (i, i + 2) and 23 (i, i + 3) NOEs, could be identified by analysis of the NOESY spectra of the denatured states, and 3J(HN, Halpha) coupling constants and 15N relaxation rates have been measured. The coupling constant and NOE data were analyzed by comparisons with theoretical predictions from a random coil polypeptide model based on amino acid specific phi,psi distributions extracted from the protein data bank. There is significant agreement between predicted and experimental NMR parameters suggesting that local conformations of the denatured states are largely determined by short-range interactions within the polypeptide chain. This result is supported by the observation that the chemical shift, coupling constant, and NOE data are little affected by whether or not the four disulfide bridge cross-links are formed in the denatured protein. The relaxation data, however, show significant differences between the oxidized and reduced protein. Analysis of the relaxation data in terms of simple dynamics models provides evidence for weak clustering of hydrophobic groups near tryptophan residues and increased barriers to motion in the more compact conformers formed when the polypeptide chain is cross-linked by the disulfide bridges. Using this information, a structural description of these denatured states is given in terms of an ensemble of conformers, which have a complex relationship between their local and global characteristics.

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Non-native states of proteins are of increasing interest because of their relevance to issues such as protein folding, translocation and stability. A framework for interpreting the wealth of experimental data for non-native states emerging from rapid advances in experimental techniques involves comparison with a "random coll' state, which possesses no structure except that inherent in the local interactions. We review here the concept of a random coil, from its global to its local properties. In particular, we focus on the description of a random coil in terms of statistical distributions in psi, phi space. We show that such a model, in combination with experimental data, provides insight into the structural properties of polypeptide chains and has significance for understanding protein folding and for molecular design.