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R M Glaeser

Publications and source records attributed to R M Glaeser.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate.

Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.

Amino Acid Substitution↗
PubMed2001

Structure of an early intermediate in the M-state phase of the bacteriorhodopsin photocycle.

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.

Bacteriorhodopsins↗
PubMed2000

Chemical and physical evidence for multiple functional steps comprising the M state of the bacteriorhodopsin photocycle.

In the photocycle of bacteriorhodopsin (bR), light-induced transfer of a proton from the Schiff base to an acceptor group located in the extracellular half of the protein, followed by reprotonation from the cytoplasmic side, are key steps in vectorial proton pumping. Between the deprotonation and reprotonation events, bR is in the M state. Diverse experiments undertaken to characterize the M state support a model in which the M state is not a static entity, but rather a progression of two or more functional substates. Structural changes occurring in the M state and in the entire photocycle of wild-type bR can be understood in the context of a model which reconciles the chloride ion-pumping phenotype of mutants D85S and D85T with the fact that bR creates a transmembrane proton-motive force.

Bacteriorhodopsins↗
PubMed1999

Review: electron crystallography: present excitement, a nod to the past, anticipating the future.

From a modest beginning with negatively stained samples of the helical T4 bacteriophage tail, electron crystallography has emerged as a powerful tool in structural biology. High-resolution density maps, interpretable in terms of an atomic structure, can be obtained from specimens prepared as well-ordered, two-dimensional crystals, and the resolution achieved with helical specimens and icosahedral viruses is approaching the same goal. A hybrid approach to determining the molecular structure of complex biological assemblies is generating great interest, in which high-resolution structures that have been determined for individual protein components are fitted into a lower resolution envelope of the large complex. With this as background, how much more can be anticipated for the future? Considerable scope still remains to improve the quality of electron microscope images. Automation of data acquisition and data processing, together with the emergence of computational speeds of 10(12) floating point operations per second or higher, will make it possible to extend high-resolution structure determination into the realm of single-particle microscopy. As a result, computational alignment of single particles, i.e., the formation of "virtual crystals," can begin to replace biochemical crystallization. Since single-particle microscopy may remain limited to "large" structures of 200 to 300 kDa or more, however, smaller proteins will continue to be studied as helical assemblies or as two-dimensional crystals. The further development of electron crystallography is thus likely to turn increasingly to the use of single particles and small regions of ordered assemblies, emphasizing more and more the potential for faster, higher throughput.

Crystallography↗
PubMed1999

Chemical bonding effects in the determination of protein structures by electron crystallography.

Scattering of electrons is affected by the distribution of valence electrons that participate in chemical bonding and thus change the electrostatic shielding of the nucleus. This effect is particularly significant for low-angle scattering. Thus, while chemical bonding effects are difficult to measure with small-unit cell materials, they can be substantial in the study of proteins by electron crystallography. This work investigates the magnitude of chemical bonding effects for a representative collection of protein fragments and a model ligand for nucleotide-binding proteins within the resolution range generally used in determining protein structures by electron crystallography. Electrostatic potentials were calculated by ab initio methods for both the test molecules and for superpositions of their free atoms. Differences in scattering amplitudes can be well over 10% in the resolution range below 5 A and are especially large in the case of ionized side chains and ligands. We conclude that the use of molecule-based scattering factors can provide a much more accurate representation of the low-resolution data obtained in electron crystallographic studies. The comparison of neutral and ionic structure factors at resolutions below 5 A can also provide a sensitive determination of charge states, important for biological function, that is not accessible from X-ray crystallographic measurements.

Crystallography↗
PubMed1998

Structural characterization of the L-to-M transition of the bacteriorhodopsin photocycle.

Structural intermediates occurring in the photocycle of wild-type bacteriorhodopsin are trapped by illuminating hydrated, glucose-embedded purple membrane at 170 K, 220 K, 230 K, and 240 K. We characterize light-induced changes in protein conformation by electron diffraction difference Fourier maps, and relate these to previous work on photocycle intermediates by infrared (FTIR) spectroscopy. Samples illuminated at 170 K are confirmed by FTIR spectroscopy to be in the L state; a difference Fourier projection map shows no structural change within the 0.35-nm resolution limit of our data. Difference maps obtained with samples illuminated at 220 K, 230 K, and 240 K, respectively, reveal a progressively larger structural response in helix F when the protein is still in the M state, as judged by the FTIR spectra. Consistent with previous structural studies, an adjustment in the position or in the degree of ordering of helix G accompanies this motion. The model of the photocycle emerging from this and previous studies is that bacteriorhodopsin experiences minimal change in protein structure until a proton is transferred from the Schiff base to Asp85. The M intermediate then undergoes a conformational evolution that opens a hydrated "half-channel," allowing the subsequent reprotonation of the Schiff base by Asp96.

Aspartic Acid↗
PubMed1997

Differences in hydration structure near hydrophobic and hydrophilic amino acids.

We use molecular dynamics to simulate recent neutron scattering experiments on aqueous solutions of N-acetyl-leucine-amide and N-acetyl-glutamine-amide, and break down the total scattering function into contributions from solute-solute, solute-water, water-water, and intramolecular correlations. We show that the shift of the main diffraction peak to smaller angle that is observed for leucine, but not for glutamine, is attributable primarily to alterations in water-water correlations relative to bulk. The perturbation of the water hydrogen-bonded network extends roughly two solvation layers from the hydrophobic side chain surface, and is characterized by a distribution of hydrogen bonded ring sizes that are more planar and are dominated by pentagons in particular than those near the hydrophilic side chain. The different structural organization of water near the hydrophobic solute that gives rise to the inward shift in the main neutron diffraction peak under ambient conditions may also provide insight into the same directional shift for pure liquid water as it is cooled and supercooled.

Amino Acids↗
PubMed1996

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.

Amides↗
PubMed1995

Crystallographic extraction and averaging of data from small image areas.

The accuracy of structure factor phases determined from electron microscope images is determined mainly by the level of statistical significance, which is limited by the low level of allowed electron exposure and by the number of identical unit cells that can be averaged. It is shown here that Fourier transforms of small image fields of purple membrane (a two-dimensional crystal consisting of bacteriorhodopsin and endogenous lipids) can be combined to provide the same quality of phases as are obtained from Fourier transforms of large image fields of the same total area. Although Fourier transforms of such small image fields are statistically significant only at lower resolution, the data from many such image fields can be averaged at the calculated positions of high-resolution reciprocal lattice points to give accurate phases. More specifically, when images of a size that can be recorded with CCD cameras are processed individually, key parameters including lattice vectors, defocus, crystal and beam tilts, and common phase origin can be accurately determined.

Crystallography, X-Ray↗
PubMed1995

The relevance of dose-fractionation in tomography of radiation-sensitive specimens.

It is commonly assumed that the number of projections required for single-axis tomography precludes its application to most beam-labile specimens. However, Hegerl and Hoppe have pointed out that the total dose required to achieve statistical significance for each voxel of a computed 3D reconstruction is the same as that required to obtain a single 2D image of that isolated voxel, at the same level of statistical significance. Thus a statistically significant 3D image can be computed from statistically insignificant projections, as long as the total dose that is distributed among these projections is high enough that it would have resulted in a statistically significant projection, if applied to only one image. We have tested this critical theorem by simulating the tomographic reconstruction of a realistic 3D model created from an electron micrograph. The simulations verify the basic conclusions of the theorem and extend its validity to the experimentally more realistic conditions of high absorption, signal-dependent noise, varying specimen contrast and missing angular range. Individual projections in the series of fractionated-dose images could be aligned by cross-correlation because they contained significant information derived from the summation of features from different depths in the structure. This latter information is generally not useful for structural interpretation prior to 3D reconstruction, owing to the complexity of most specimens investigated by single-axis tomography. These results demonstrate that it is feasible to use single-axis tomography with soft X-ray and electron microscopy of frozen-hydrated specimens.

Centrioles↗
PubMed1994

Overview of significant challenges in molecular biology amenable to computational methods.

Many challenging but significant opportunities exist for the development of theoretical approaches in modern Cell and Molecular Biology. The creation of data bases which contain extremely large amounts of information has proven to be an unexpectedly important facto-tin gaining acceptance and respectability for theoretical work that builds on nothing more than what is in the data base itself, such as theoretical work involving the analysis of known protein structures, or the development of more powerful homology searches. Other opportunities, not yet accepted by a broad community, involve work on complex networks (metabolic, genetic, immunologic and neural networks) and work on the "physics of how things work." The DOE National Laboratory System represents the ideal institution that would be well suited to the role of being an "incubator" for the creation of a theoretical and computational discipline within modern biology.

Adenosine Triphosphatases↗
PubMed1994

Specimen flatness of glucose-embedded biological materials for electron crystallography is affected significantly by the choice of carbon evaporation stock.

Imperfect specimen flatness can be a significant limitation in the application of electron crystallography to high-resolution structure analysis of biological macromolecules. We now report that the choice of solid carbon stock that is used to make evaporated carbon films can have a very great effect on the preparation of flat specimens of glucose-embedded purple membrane. The degree of purity of the carbon does not seem to be the controlling factor, and other likely factors such as the type of mica used as a substrate, the evaporation apparatus used (and its limiting vacuum), and the use of a continuous versus an interrupted evaporation protocol do not have a discernible influence. The physical or chemical basis for the observed differences in specimen flatness is still unknown; however, the important conclusion that we can communicate at this point is that the choice of evaporating material does have a major effect on the flatness of purple membrane, the specimen used here. The implication is that different sources of carbon stock should be tried whenever difficulty is encountered in the preparation of suitably flat specimens of biological macromolecules.

Crystallography↗
PubMed1994

Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin.

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.

Bacteriorhodopsins↗
PubMed1994

The bacteriorhodopsin photocycle: direct structural study of two substrates of the M-intermediate.

Changes in protein structure that occur during the formation of the M photointermediate of bacteriorhodopsin can be directly visualized by electron diffraction techniques. A modified preparation technique for glucose-embedded crystals was employed to ensure sufficient hydration of the crystals, which was needed for the formation of the M intermediate at low temperature. Samples containing a high percentage of the M intermediate were trapped by rapidly cooling the crystals with liquid nitrogen after illumination with filtered green light at 240 and 260 K, respectively. Difference Fourier projection maps are presented for the M intermediates formed at these two temperatures. The diffraction data clearly show that statistically significant structural changes occur upon formation of the M intermediate at 240 K and then further upon formation of the second specimen that is produced at 260 K.

Bacteriorhodopsins↗
PubMed1993

High-resolution electron crystallography of protein molecules.

Electron diffraction data and high-resolution images can now be used to obtain accurate, three-dimensional density maps of biological macromolecules. These density maps can be interpreted by building an atomic-resolution model of the structure into the experimental density. The Cowley-Moodie formalism of dynamical diffraction theory has been used to validate the use of kinematic diffraction theory (strictly, the weak phase object approximation) in producing such 3D density maps. Further improvements in the preparation of very flat (planar) specimens and in the retention of diffraction to a resolution of 0.2 nm or better could result in electron crystallography becoming as important a technique as X-ray crystallography currently is for the field of structural molecular biology.

Crystallography↗
PubMed1993

Glucose alone does not completely hydrate bacteriorhodopsin in glucose-embedded purple membrane.

Glucose embedding is a simple and highly effective method for preparing biological macromolecules for high-resolution electron microscopy. The investigation of conditions that can trap the M-state intermediate in the bacteriorhodopsin (bR) photocycle has revealed, however, that when glucose-embedded bR is prepared at ambient humidity, it does not fully retain the capability to execute a proper photocycle. However, 'native' photocycle properties are returned after glucose-embedded samples are equilibrated at 81% relative humidity. Equilibration at relative humidities significantly higher than 81% causes glucose to dissolve in its own water of hydration, resulting in samples that may be too thick to be suitable for electron microscopy. The results obtained with bR indicate that caution should be taken with other biological specimens, and it cannot be assumed that glucose-embedded biological macromolecules retain completely their native, hydrated structure, even when high-resolution electron diffraction patterns are obtained. Equilibration of such samples at high humidity may generally be a worthwhile precaution when using the glucose-embedding technique.

Bacteriorhodopsins↗
PubMed1992

Specimen flatness of thin crystalline arrays: influence of the substrate.

The extreme degree of specimen flatness (i.e. planarity) required for high-resolution electron diffraction and electron microscopy at high tilt angles cannot be realized with thin, sheet-like crystals of biological macromolecules, just on the basis of the intrinsic stiffness of the specimen itself. In an effort to improve the rate of success at which suitably flat specimens are prepared, this paper analyzes several different factors that can either limit or enhance the specimen flatness. If specimens are adsorbed (by attractive forces) to a support film, such as evaporated carbon, which itself is not flat to atomic dimensions, quantitative calculations show that it is quite likely that the specimen will be too wrinkled to be used for high-resolution studies. Adsorption to an air-water interface is more likely to result in the necessary degree of flatness. Repulsive interactions, which might be used to "sandwich" a specimen between two interfaces, are estimated to be too "soft", i.e. too long-range in character, to be effective. Finally, if only one edge of a specimen sticks firmly to a substrate, then surface tension forces can pull the specimen taut over the surface of the substrate, so that the specimen itself can be more flat than the surface of the substrate upon which it is deposited. A second, important consideration in many studies is the fact that cooling the specimen to low temperature can result in specimen wrinkling, because of the fact that the biological crystal has a much larger coefficient of thermal expansion than that of the evaporated carbon film. In this case one expects that cooling-induced wrinkling might be reduced by using a metal support grid which has a smaller thermal coefficient than that of the carbon film. The validity of this qualitative idea is supported by experiments which show that cooling-induced wrinkling of glucose-embedded purple membrane can be prevented if molybdenum grids are used rather than copper.

Artifacts↗