Understanding how monomeric proteins fold under in vitro conditions is crucial to describing their functions in the cellular context. Significant advances in theory and experiments have resulted in a conceptual framework for describing the folding mechanisms of globular proteins. The sizes of proteins in the denatured and folded states, cooperativity of the folding transition, dispersions in the melting temperatures at the residue level, and timescales of folding are, to a large extent, determined by N, the number of residues. The intricate details of folding as a function of denaturant concentration can be predicted by using a novel coarse-grained molecular transfer model. By watching one molecule fold at a time, using single-molecule methods, investigators have established the validity of the theoretically anticipated heterogeneity in the folding routes and the N-dependent timescales for the three stages in the approach to the native state. Despite the successes of theory, of which only a few examples are documented here, we conclude that much remains to be done to solve the protein folding problem in the broadest sense.

We investigate the kinetics of loop formation in ideal flexible polymer chains (the Rouse model), and polymers in good and poor solvents. We show for the Rouse model, using a modification of the theory of Szabo, Schulten, and Schulten, that the time scale for cyclization is tau(c) approximately tau(0)N(2) (where tau(0) is a microscopic time scale and N is the number of monomers), provided the coupling between the relaxation dynamics of the end-to-end vector and the looping dynamics is taken into account. The resulting analytic expression fits the simulation results accurately when a, the capture radius for contact formation, exceeds b, the average distance between two connected beads. Simulations also show that when a < b, tau(c) approximately N(alpha)(tau), where 1.5 < alpha(tau) < or = 2 in the range 7 < N < 200 used in the simulations. By using a diffusion coefficient that is dependent on the length scales a and b (with a < b), which captures the two-stage mechanism by which looping occurs when a < b, we obtain an analytic expression for tauc that fits the simulation results well. The kinetics of contact formation between the ends of the chain are profoundly effected when interactions between monomers are taken into account. Remarkably, for N < 100, the values of tau(c) decrease by more than 2 orders of magnitude when the solvent quality changes from good to poor. Fits of the simulation data for tau(c) to a power law in N (tau(c) approximately N(alpha)(tau)) show that alpha(tau) varies from about 2.4 in a good solvent to about 1.0 in poor solvents. The effective exponent alpha(tau) decreases as the strength of the attractive monomer-monomer interactions increases. Loop formation in poor solvents, in which the polymer adopts dense, compact globular conformations, occurs by a reptation-like mechanism of the ends of the chain. The time for contact formation between beads that are interior to the chain in good solvents changes nonmonotonically as the loop length varies. In contrast, the variation in interior loop closure time is monotonic in poor solvents. The implications of our results for contact formation in polypeptide chains, RNA, and single-stranded DNA are briefly outlined.

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SynopsisA new facility for microdiffraction strain measurements and microfluorescence mapping has been developed at the Advanced Light Source. Details of the mechanics and performance of the beamline and endstation will be given. AbstractA new facility for microdiffraction strain measurements and microfluorescence mapping has been built on beamline 12.3.2 at the Advanced Light Source (ALS) of the Lawrence Berkeley National Laboratory (LBNL).This beamline benefits from the hard x-radiation generated by a 6 Tesla superconducting bending magnet (superbend). This provides a hard x-ray spectrum from 5 keV to 22 keV and a flux within a 1 µm spot of ~ 5 · 10 9 photons per seconds (0.1% bandwidth at 8 keV). The radiation is relayed from the superbend source to a focus in the experimental hutch by a toroidal mirror. The focus spot is tailored by two pairs of adjustable slits, which serve as secondary source point. Inside the lead hutch, a pair of Kirkpatrick-Baez (KB) mirrors placed in a 2 vacuum tank re-focuses the secondary slit source onto the sample position. A new KB-bending mechanism with active temperature stabilization allows for more reproducible and stable mirror bending and thus mirrorfocusing. Focus spots around 1 µm are routinely achieved and allow a variety of experiments, which have in common the need of spatial resolution. The effective spatial resolution (~0.2 µm) is limited by a convolution of beam size, scan-stage resolution and stage stability. A 4-bounce monochromator consisting of 2 channel-cut Si(111) crystals placed between the secondary source and KB-mirrors allows for easy changes between whitebeam and monochromatic experiments while maintaining a fixed beam position. High resolution stage scans are performed while recording a fluorescence emission signal or an x-ray diffraction signal coming from either a monochromatic or a white focused beam. The former allows for elemental mapping, whereas the latter is used to produce 2-dimensional maps of crystal-phases,-orientation, -texture and -strain/stress. Typically achieved strain resolution is in the order of 5 · 10 -5 strain units. Accurate sample positioning in the x-ray focus spot is achieved with a commercial laser-triangulation unit. A Si-drift detector serves as a high-energy-resolution (~150 eV FWHM) fluorescence detector. Fluorescence scans can be collected in continuous scan mode with up to 300 pixels per second scan-speed. A CCD area detector is utilized as diffraction detector. Diffraction can be performed in reflecting or transmitting geometry. Diffraction data are processed using XMAS, an in-house written software package for Laue and monochromatic microdiffraction analysis.