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L M Rice

Publications and source records attributed to L M Rice.

At least 19 recordsLinked to original sources
PubMed2000

Single-wavelength anomalous diffraction phasing revisited.

Multiwavelength anomalous diffraction (MAD) phasing has become a routinely used tool for determining new macromolecular structures. The MAD method has stringent data-collection requirements, typically necessitating radiation-resistant crystals and access to a tunable synchrotron beamline. In cases where synchrotron time, monochromator tunability or radiation damage is a concern or where high-throughput structure determination is desired, phasing methods capable of producing interpretable electron-density maps from less data become attractive alternatives to MAD. The increasing availability of tunable synchrotron data-collection facilities prompted the authors to revisit single-wavelength anomalous diffraction (SAD) phasing used in conjunction with a phase-ambiguity resolving method such as solvent flattening. The anomalous diffraction from seven different selenomethionine-labelled protein crystals has been analysed and it is shown that in conjunction with solvent flattening, diffraction data from the peak anomalous wavelength alone can produce interpretable electron-density maps of comparable quality to those resulting from full MAD phasing. Single-wavelength anomalous diffraction (SAD) phasing can therefore be a time-efficient alternative to MAD. The data also show that radiation damage can have a significant effect on the quality of SAD/MAD diffraction data. These results may be useful in the design of optimal strategies for collection of the diffraction data.

Models, Molecular↗
PubMed1999

Long-term variations in cyclic light intensity and dietary vitamin A intake modulate lipofuscin content of the retinal pigment epithelium.

Experiments were conducted to determine whether the intensity of cyclic light exposure to the retina over a long period of time affects retinoid-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE). Albino rats were maintained from weaning on diets either containing (+A) or lacking (-A) retinyl palmitate, which can be metabolized to the retinoids involved in the visual cycle. Animals in each dietary group were divided between bright (L) and dim (D) cyclic light treatments. Thus, the experiments employed the following four treatment groups: +A/D, +A/L, -A/D, and -A/L. After 6, 12, and 15 months from the start of the treatments, animals in each group were killed for quantitative determination of: 1) retinal photoreceptor densities; 2) RPE lipofuscin content; and 3) RPE lipofuscin fluorescence intensity. Animals in the L groups had a lower volume of RPE lipofuscin than those in the D groups fed the same diet. Among the -A rats, this reduced lipofuscin volume could be attributed to a light-enhanced depletion of vitamin A from the retina and an accompanying loss of photoreceptor cells. In the +A animals, however, there were no differences in photoreceptor densities between the D and L groups. In the -A rats, the volume of RPE lipofuscin decreased between 6 and 15 months of age, whereas it increased in the +A animals. In contrast, lipofuscin fluorescence intensity increased between 6 and 15 months of age in all four treatment groups. However, in the +A rats, the fluorescence intensity was lower in the L than in the D group at all three ages. In the -A groups, light level had no effect on lipofuscin fluorescence intensity. At all three ages, fluorescence intensity was lower in the -A animals than in +A rats. Thus, at light intensities below those that induce acute retinal degeneration, long-term exposure to higher intensity light inhibits the age-related increase in RPE lipofuscin volume. A decrease in the volume of RPE lipofuscin after the retina is depleted of vitamin A suggests that lipofuscin is turned over, and that RPE lipofuscin content is determined by a balance between the rates at which lipofuscin is formed and at which it is eliminated from the RPE. An age-related increase in lipofuscin-specific fluorescence intensity after vitamin A depletion from the retina suggests that lipofuscin fluorophores may continue to form slowly from retinoids that have been modified such that they can no longer enter the visual cycle.

Animals↗
PubMed1999

Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel.

The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.

Amino Acid Sequence↗
PubMed1999

Crystal structure of the vesicular transport protein Sec17: implications for SNAP function in SNARE complex disassembly.

SNAP proteins play an essential role in membrane trafficking in eukaryotic cells. They activate and recycle SNARE proteins by serving as adaptors between SNAREs and the cytosolic chaperone NSF. We have determined the crystal structure of Sec17, the yeast homolog of alpha-SNAP, to 2.9 A resolution. Sec17 is composed of an N-terminal twisted sheet of alpha-helical hairpins and a C-terminal alpha-helical bundle. The N-terminal sheet has local similarity to the tetratricopeptide repeats from protein phosphatase 5 but has a different overall twist. Sec17 also shares structural features with HEAT and clathrin heavy chain repeats. Possible models of SNAP:SNARE binding suggest that SNAPs may function as lever arms, transmitting forces generated by conformational changes in NSF/Sec18 to drive disassembly of SNARE complexes.

Amino Acid Sequence↗
PubMed1999

Folding intermediates of SNARE complex assembly.

SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.

Circular Dichroism↗
PubMed1999

Reversible accumulation of lipofuscin-like inclusions in the retinal pigment epithelium.

PURPOSE: The amounts of autofluorescent lysosomal storage bodies, known as lipofuscin, increase during senescence in the retinal pigment epithelia (RPEs) of mammalian eyes. This increase in lipofuscin content may result from a failure of the RPE to dispose of any lipofuscin constituents once they have formed. Alternatively, the RPE may eliminate lipofuscin but at a rate insufficient to prevent its accumulation. Experiments were conducted to distinguish between these two possibilities. METHODS: Albino rats were given intravitreal injections of the protease inhibitor leupeptin, which induces a rapid accumulation of lipofuscin-like inclusions in the RPE. The amount of these inclusions in the RPE was monitored as a function of time after the leupeptin treatment with quantitative ultrastructural analysis. In addition, the intensity of lipofuscin-specific fluorescence in the RPE was monitored over the same time period with the use of quantitative microfluorometry. These parameters were also followed in untreated control eyes of age-matched animals. RESULTS: A single leupeptin injection resulted in a rapid massive accumulation of electron-dense inclusion bodies in the RPE. These inclusions appeared to be derived primarily from phagocytosed photoreceptor outer segments. Accompanying the accumulation of these inclusions was a significant increase in lipofuscin-specific fluorescence in the RPE. Over a 12-week period after the leupeptin treatment, the amounts of inclusion material and the fluorescence intensities returned to normal levels. CONCLUSIONS: These findings suggest that the age-related increase in RPE lipofuscin content results from an imbalance in the rates of lipofuscin formation and disposal rather than from a complete absence of a disposal mechanism. The results imply that turnover of lipofuscin constituents may be rapid relative to the animals' life span. Thus, it may be possible to slow or reverse the age-related increase in RPE lipofuscin content by either inhibiting the processes involved in lipofuscin formation or enhancing the disposal processes.

Aging↗
PubMed1998

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Computer Simulation↗
PubMed1997

Modulation of nerve growth factor internalization by direct interaction between p75 and TrkA receptors.

While the central role played by TrkA in nerve growth factor (NGF) signalling has been established by dissecting its signal transduction pathways, insight into the mechanism of action of p75LNR, the low-affinity neurotrophin receptor, has only recently been achieved. The relative contribution of p75LNR and TrkA to the constitution of high-affinity receptors for NGF and, similarly, with TrkB an TrkC to the formation of those for other neurotrophins, is presently under debate. Some form of collaboration in mediating neurotrophin activities has been observed between the Trk and p75LNR receptors, but only recent indirect evidence indicates a molecular interaction. In the present work, we have ectopically coexpressed p75LNR and TrkA in Sf9 insect cells by using baculovirus vectors, and show a direct association between the two NGF receptors. In addition, we show that the intracellular and extracellular domains of both receptors contribute to this interaction. Finally, we demonstrate that NGF becomes endocytosed in TrkA-expressing cells but not in p75LNR-expressing cells, and that such function can be modulated by the presence of the intracellular domain of p75LNR receptor.

Animals↗
PubMed1997

Formation of a yeast SNARE complex is accompanied by significant structural changes.

The evolutionarily conserved SNARE (SNAP receptor) proteins and their complexes are key players in the docking and fusion of secretory vesicles with their target membrane. Biophysical techniques were used to characterize structural and energetic properties of the cytoplasmic domains of the yeast SNAREs Snc1 and Sso1, of the SNAP-25-like domain of Sec9, and of the Sso1:Sec9 and Sso1:Sec9:Snc1 complexes. Individually, all three SNAREs are monomeric; Sso1 shows significant secondary structure while Snc1 and Sec9 are largely unstructured. Ternary SNARE complex formation (KD <50 nM) is accompanied by a more than two-fold increase in secondary structure. This binding induced structure, the large increase in thermal stability, and the self-association of the ternary complex represent conserved properties of SNAREs that are probably important in vesicle docking and fusion.

Base Sequence↗
PubMed1997

Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9.

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

Amino Acid Sequence↗
PubMed1997

Torsion-angle molecular dynamics as a new efficient tool for NMR structure calculation.

Molecular dynamics in torsion-angle space was applied to nuclear magnetic resonance structure calculation using nuclear Overhauser effect-derived distances and J-coupling-constant-derived dihedral angle restraints. Compared to two other commonly used algorithms, molecular dynamics in Cartesian space and metric-matrix geometry combined with Cartesian molecular dynamics, the method shows increased computational efficiency and success rate for large proteins, and it shows a dramatically increased radius of convergence for DNA. The torsion-angle molecular dynamics algorithm starts from an extended strand conformation and proceeds in four stages: high-temperature torsion-angle molecular dynamics, slow-cooling torsion-angle molecular dynamics, Cartesian molecular dynamics, and minimization. Tests were carried out using experimental NMR data for protein G, interleukin-8, villin 14T, and a 12 base-pair duplex of DNA, and simulated NMR data for bovine pancreatic trypsin inhibitor. For villin 14T , a monomer consisting of 126 residues, structure determination by torsion-angle molecular dynamics has a success rate of 85%, a more than twofold improvement over other methods. In the case of the 12 base-pair DNA duplex, torsion-angle molecular dynamics had a success rate of 52% while Cartesian molecular dynamics and metric-matrix distance geometry always failed.

Algorithms↗
PubMed1997

Crystal structure of the two RNA binding domains of human hnRNP A1 at 1.75 A resolution.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant eukaryotic nuclear RNA binding protein. A1 is involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm and may modulate splice site selection. The crystal structure of A1(RBD1,2) reveals two independently-folded RNA binding domains (RBDs) connected by a flexible linker. Both RBDs are structurally homologous to the U1A(RBD1), and have their RNA binding platforms oriented in an anti-parallel fashion. The anti-parallel arrangement of the A1 RNA binding platforms suggests mechanisms for RNA condensation and ways of bringing together distant RNA sequences for RNA metabolism such as splicing or transport.

Binding Sites↗
PubMed1996

Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes.

During the aging process the retinal pigment epithelium (RPE) accumulates autofluorescent lysosomal storage bodies (lipofuscin). Data from previous studies led to the hypothesis that at least one of the fluorescent components of RPE lipofuscin is formed by reaction of vitamin A aldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All-trans retinaldehyde was incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes. Liposomes were made with two different lipid compositions. One type of liposome consisted of a mixture of lipids, including phosphatidylcholine (PC), none of which contained a primary amine. The other liposome type was identical in composition accept that some of the PC was replaced with an equimolar amount of phosphatidylethanolamine (PE). After incubation of the samples, aliquots were examined with fluorescence microscopy to assess whether any lipofuscin-like fluorescence had developed. Lipids were extracted from additional aliquots of the samples and analyzed with thin layer chromatography. Photoreceptor outer segments incubated with retinaldehyde developed an intense golden yellow fluorescent emission when illuminated with 395-440 nm light. Similar fluorescence developed in the liposomes containing PE, whereas the liposomes lacking PE or any other primary amine did not develop any detectable fluorescence. The development of fluorescence in the samples in situ correlated with the appearance of an orange colored component in the lipid extracts that displayed a weak red emission upon ultraviolet light illumination. Incorporation of this component into liposomes resulted in the appearance of a golden yellow fluorescent emission. The results of these experiments suggest that retinal, generated during visual pigment bleaching can react with PE in the photoreceptor outer segments to form a fluorophore, a derivative of which subsequently accumulates in RPE lipofuscin. An RPE lipofuscin fluorophore was previously shown to be identical to a reaction product of retinal and ethanolamine. This fluorophore is probably derived from the reaction product of outer segment PE and retinal.

Animals↗
PubMed1995

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation.

Amino Acid Sequence↗
PubMed1994

Torsion angle dynamics: reduced variable conformational sampling enhances crystallographic structure refinement.

A reduced variable conformational sampling strategy for macromolecules based on molecular dynamics in torsion angle space is evaluated using crystallographic refinement as a prototypical search problem. Bae and Haug's algorithm for constrained dynamics [Bae, D.S., Haug, E.J. A recursive formulation for constrained mechanical system dynamics. Mech. Struct. Mach. 15:359-382, 1987], originally developed for robotics, was used. Their formulation solves the equations of motion exactly for arbitrary holonomic constraints, and hence differs from commonly used approximation algorithms. It uses gradients calculated in Cartesian coordinates, and thus also differs from internal coordinate formulations. Molecular dynamics can be carried out at significantly higher temperatures due to the elimination of the high frequency bond and angle vibrations. The sampling strategy presented here combines high temperature torsion angle dynamics with repeated trajectories using different initial velocities. The best solutions can be identified by the free R value, or the R value if experimental phase information is appropriately included in the refinement. Applications to crystallographic refinement. Applications to crystallographic refinement show a significantly increased radius of convergence over conventional techniques. For a test system with diffraction data to 2 A resolution, slow-cooling protocols fail to converge if the backbone atom root mean square (rms) coordinate deviation from the crystal structure is greater than 1.25 A, but torsion angle refinement can correct backbone atom rms coordinate deviations up to approximately 1.7 A.

Algorithms↗