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F M Richards

Publications and source records attributed to F M Richards.

At least 109 records · Page 6Linked to original sources
PubMed1985

1H-15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type.

Ten samples of Escherichia coli thioredoxin were individually isotopically enriched by residue type via growth of an appropriate auxotrophic strain on media supplemented with one 2H, 15N-enriched amino acid. 1H observe-heteronuclear decoupling experiments were conducted on these samples making use of the 95-Hz 1H-15N amide J1 coupling. Subtraction of near-resonance from off-resonance 15N decoupled spectra generated difference patterns corresponding only to protons directly bonded to 15N nuclei. For the ten different enriched residue types observed to date, every labeled position (60) has been observed as a resolved resonance. The spectral dispersion in both the 1H and the 15N dimensions was roughly 1500 Hz (at 500-MHz field strength) with rather little apparent dependence on residue type. With the exception of the glycine-enriched sample, the range of the J1 coupling constants was not much greater than the precision of the measurements (1.5-2.0 Hz). However, for the glycine residues the J1 amide coupling values varied over a range of 10 Hz.

Bacterial Proteins↗
PubMed1985

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle X-ray scattering.

We have used solution small-angle X-ray scattering to characterize bovine brain calmodulin in the presence and absence of calcium. In the presence of calcium, calmodulin exists in solution as an elongated molecule with a radius of gyration of 21.5 A and a maximum vector length of approximately 62 A. These values are consistent with the dimensions recently determined for the crystal form of rat testis calmodulin. In the absence of calcium, the calmodulin molecule is shorter, the radius of gyration decreases to 20.6 A, and the maximum vector length decreases to approximately 58 A. This change in dimensions is consistent with an overall contraction of the protein through movement of the two lobes closer to each other upon removal of calcium from calmodulin.

Animals↗
PubMed1985

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis.

Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated 3H leucine from the digested membranes. During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.

Amino Acid Sequence↗
PubMed1984

Insertion of apocytochrome c into lipid vesicles.

Apocytochrome c (cytochrome c without the heme) is synthesized in the cell cytoplasm without a cleaved signal sequence, then transported across the outer mitochondrial membrane. We have studied the interaction of apocytochrome c with lipid vesicles as a model for understanding protein translocation across membranes. Apocytochrome c (but not holocytochrome c) that has been incubated with vesicles at 37 degrees C in 0.2 M NaCl binds to the vesicles. Under these conditions, as well as upon incubation with detergent or at high protein concentrations, all the added protein remains partly accessible to externally added protease, but a COOH-terminal fragment of some of the protein molecules becomes protected against digestion. When apocytochrome c is added to azolectin vesicles with internally trapped proteases, most of the added protein can be digested, even in the presence of a large excess of protease inhibitor external to the vesicles. Thus, in spite of a lack of nonpolar stretches in its amino acid sequence, apocytochrome c is capable of binding to and inserting into lipid membranes. In this model system, transport may be driven by trapping of protease-digested apocytochrome c on one side of the membrane.

Amino Acid Sequence↗
PubMed1983

Dimerization by colicin E3 in the absence of immunity protein.

Using small angle x-ray scattering from solutions of colicin E3-immunity protein complex and the separated proteins, we have measured the radii of gyration of each species. We find that the complex is an elongated molecule with a radius of gyration of 35.5 +/- 0.7 A. Immunity protein is more compact with a radius of gyration of 13.4 +/- 0.1 A. Upon removal of immunity protein from the complex, colicin E3 (form minus immunity protein) undergoes further elongation and dimerizes, such that its radius of gyration increases to 75.6 +/- 3.1 A. Dimerization is not reversed by simple addition of a stoichiometric amount of immunity protein to the E3 solution, even though this is sufficient to block in vitro activity. We suggest that the elongation, and perhaps dimer formation, occurs in vivo, concomitant with membrane translocation.

Bacterial Proteins↗
PubMed1983

The pH dependence of hydrogen exchange in proteins.

The static accessibility modified discrete charge model for electrostatic interactions in proteins is extended to the prediction of the pH dependence of hydrogen exchange reactions. The exchange rate profiles of buried amide protons are shown to follow the calculated pH dependence of the electrostatic component of protein stability. Rate profiles are calculated for individual buried amide protons in ribonuclease S and bovine pancreatic trypsin inhibitor. The electrostatic free energy of stabilization of the protein and the energy required to bring the catalytic ion to an exchange site are expressed as an apparent, pH-dependent contribution to the activation energy. Changes in the electrostatic stabilization of the proteins affect the calculated exchange rate for buried amide protons by more than 1000, while local field effects raise or lower the predicted exchange rates by less than 100. The pH dependence of exchangeable protons at the protein surface, such as the C-2 imidazole protons, is shown to follow the estimated energy required to introduce the catalytic ion at the exchange site. These calculations are discussed in terms of current models for proton exchange which incorporate the dynamic nature of the structure to explain exchange data from the interior of a protein.

Hydrogen-Ion Concentration↗
PubMed1983

Electrostatic orientation during electron transfer between flavodoxin and cytochrome c.

Various studies have shown that reaction rates between reversibly binding electron transfer proteins depend strongly on solution ionic strength. These observations suggest that intermolecular electrostatic interactions are important in facilitating the formation of a productive reaction complex. A recently examined system involves the reduction of vertebrate cytochrome c by bacterial flavodoxin. Although this is a nonphysiological reaction, it proceeds with rates typical for natural partners and is similarly inhibited at high ionic strengths. Here we describe computational studies which examine the role of electrostatics in the formation of a putative reaction complex between flavodoxin and cytochrome c. The results suggest that electrostatic interactions preorient the molecules before they make physical contact, facilitating the formation of an optimal reaction complex.

Chemical Phenomena↗
PubMed1982

A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-A resolution.

The crystal structure of alamethicin in nonaqueous solvent has been determined, and refined at 1.5-A resolution. The molecular conformation of the three crystallographically independent molecules is largely alpha-helical with a bend in the helix axis at an internal proline residue. The helix structure is highly amphipathic as most of the solvent-accessible polar atoms lie on a narrow strip of surface parallel to the helix axis. Molecular models for the voltage-gated ion channel, with n-fold symmetry and based on the molecular conformations observed in the crystal, are characterized by strong surface complementarity, a hydrophilic interior and a hydrophobic exterior. The channel structures are stabilized by a hydrated annulus of hydrogen-bonded glutamine residues which produce the greatest restriction in the channel diameter.

Alamethicin↗
PubMed1982

Anion binding and pH-dependent electrostatic effects in ribonuclease.

The solvent-accessibility-modified, Tanford-Kirkwood, discrete charge model for electrostatic effects is applied to both ribonuclease A and ribonuclease S. The behavior of individual titratable sites and the pH-dependent free energy of denaturation are correctly predicted. The use of the solvent-accessibility factor in reducing charge-site interactions introduces a higher Coulombic shielding for solvent-exposed sites. This shielding is interpreted as a higher local strength or alternatively a higher effective dielectric constant. Specific anion binding sites are determined by locating areas of high positive electrostatic potential at the protein solvent interface. The potential and thus the anion affinity of a given site are calculated and shown to vary with the pH-dependent charge array. pH-dependent anion binding constants are calculated for the ribonuclease S active site. These binding constants and the predicted response of the active-site histidine pK1/2 values to anion binding are shown to agree with experimental determinations.

Anions↗
PubMed1982

Increase in apparent compressibility of cytochrome c upon oxidation.

The apparent molal adiabatic compressibilities of ferri- and ferrocytochrome c have been determined from measurements of density and sound velocity. The values found were +2.99 X 10(-8) and -2.40 X 10(-8) cm5 mol-1 dyne-1 for the ferri and ferro forms, respectively. Experiments were performed on identical solutions containing either the oxidized or reduced form of protein. Solutions of ferricytochrome c were found to have significantly greater adiabatic compressibility than equivalent solutions of ferrocytochrome c at 25 degrees C and pH 7.15. The remarkable similarity of the three-dimensional structures of the ferri and ferro proteins [Takano, T. & Dickerson, R.E. (1980) Proc. Natl. Acad. Sci. USA 77, 6371-6375] strongly suggests that this difference in compressibility is due to an increase in volume fluctuations within ferricytochrome c relative to the ferro form rather than a change in equilibrium structure or hydration. Such a difference in the dynamic properties of the structures is consistent with both the crystallographic thermal B factors and the observed increase in amide hydrogen exchange kinetics when ferrocytochrome c is oxidized. The relative magnitude of the root mean square volume fluctuations is approximated from an ideal solution treatment of the compressibility data and yields a ratio of delta Vrms (ferri cyt c)/ delta Vrms (ferro cyt c) = 1.3.

Animals↗