Evaluation of macromolecular electron-density map quality using the correlation of local r.m.s. density

The correlation of local r.m.s. density is shown to be a good measure of the presence of distinct solvent and macromolecule regions in macromolecular electron-density maps

Discrimination of solvent from protein regions in native Fouriers as a means of evaluating heavy-atom solutions in the MIR and MAD methods

The presence of distinct regions of high and low density variation in electron-density maps is found to be a good indicator of the correctness of a heavy-atom solution in the MIR and MAD methods

Rate-window methods and myoglobin dynamics

The problem of protein dynamics is introduced and its significance explained. Properties of the oxygen-storage protein myoglobin (Mb) as a model system for dynamics studies are discussed. Special attention is paid to Mb's physiological role, and the basic quantities that describe the protein's function. Background on ligand binding experiments in Mb is reviewed and appropriate mathematical models established. The specific goal of this work is to determine as many as possible of the model parameters (pre-exponentials and activation enthalpies of intrinsic rate coefficients), with a view towards calculation of one functionally important quantity, the CO affinity at physiological temperatures.Relaxation spectroscopy is a powerful means for studying dynamics. Different perturbations and observables are considered, and two kinetic methods are introduced: temperature-derivative spectroscopy (TDS), a non-isothermal technique that measures the derivative of a population with respect to temperature; and deep-level transient spectroscopy (DLTS), an isothermal technique that determines the behavior of a small range of rate coefficients as a function of temperature. These "rate-window" methods are shown to be widely applicable and may prove highly advantageous in difficult measurements such as kinetic X-ray crystallography.TDS and DLTS were used to study the rebinding of CO to sperm whale Mb after photolysis. FTIR measurements of geminate rebinding in the CO-stretch bands show distributed activation enthalpies with different distributions for each band, crossing between two bands that correspond to photolyzed ligands, and kinetic hole-burning. The distributions of activation enthalpies are well described by gaussians; the results match and complement those of traditional multi-rate methods. Further experiments determined the barriers to entry to and escape from the heme pocket for two of the bands. Information about barriers to different kinds of conformational changes were also obtained.The kinetic differences among different protein conformations provide a mechanism by which the affinity of Mb might be modified in response to physiological demands. It is shown that this effect could be larger than that of the R- to T-state change in hemoglobin. Findings from the physiological and biochemical literature consistent with this possibility are pointed out, and specific tests are proposed.U of I OnlyETDs are only available to UIUC Users without author permissio

Out of the blue - the photocycle of the photoactive yellow protein

A first real glance at the structural, spectral and temporal interplay that constitutes the photocycle of the photoactive yellow protein (PYP) has been obtained from a combination of time-resolved crystallography with mutational analysis and spectroscopic studies.<BR><BR>As ones plane slips down into San Francisco airport, an alert traveler might notice a multicolored patchwork of drying basins at the margins of the bay below. These ponds, which are used to dry seawater for salt production, range in hue from muddy brown to brilliant red and even light purple, depending on the microorganisms living there. Among these are Ectothiorhodospira halophila — purple bacteria that are not only attracted to light to do an honest days photosynthesis, but also swim away from the damaging effects of blue light. The photosensors that initiate this negative phototactic response are the photoactive yellow proteins (PYPs) or Xanthopsins, so called for their brilliant yellow color. (PYPs should not be confused with the old yellow enzyme, a flavoprotein for which the structure was also solved recently.) Photoactive proteins are excellent systems in which to investigate the functional consequences of protein dynamics, because the chromophore (light-absorbing group) acts as an inherent trigger that permits synchronization of events on timescales as short as tens of femtoseconds. Also, their remarkable optical properties — their absorption spectra are broad, intense and commonly shift by about 50 nm upon absorption of a single photon — offer potential technological applications to nonlinear optics and optical computing. At present, only a handful of photoactive proteins have been characterized, and far fewer have been crystallized. PYP from E. halophila, despite its relatively recent discovery [5], is in many ways the most attractive model system because it is small (14kDa) and soluble, and its high-resolution (1.4 Å) structure has been determined

Crystal structures of myoglobin-ligand complexes at near-atomic resolution

AbstractWe have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2Å. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25Å root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18±3° with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO2 seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not

Structure of a ligand-binding intermediate in wild-type carbonmonoxy myoglobin

Small molecules such as NO, O2, CO or H2 are important biological ligands that bind to metalloproteins to function crucially in processes such as signal transduction, respiration and catalysis. A key issue for understanding the regulation of reaction mechanisms in these systems is whether ligands gain access to the binding sites through specific channels and docking sites, or by random diffusion through the protein matrix. A model system for studying this issue is myoglobin, a simple haem protein. Myoglobin has been studied extensively by spectroscopy, crystallography, computation and theory. It serves as an aid to oxygen diffusion but also binds carbon monoxide, a byproduct of endogenous haem catabolism. Molecular dynamics simulations, random mutagenesis and flash photolysis studies indicate that ligand migration occurs through a limited number of pathways involving docking sites. Here we report the 1.4 ? resolution crystal structure of a ligand-binding intermediate in carbonmonoxy myoglobin that may have far-reaching implications for understanding the dynamics of ligand binding and catalysis