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S W Englander

Publications and source records attributed to S W Englander.

At least 91 records · Page 5Linked to original sources
PubMed1983

Normal mode paths for hydrogen exchange in the peptide ferrichrome.

Possible paths for exposure to solvent and hydrogen exchange of the amide protons of ferrichrome, a cyclic hexapeptide, are examined. The paths are obtained from calculations of the vibrational normal modes of ferrichrome and correspond to low energy atomic displacements away from the local minimum in the multidimensional conformational space of the molecule. Exposure of exchangeable groups along the normal modes was determined by using the solvent accessible surface area algorithm of Lee and Richards. Three of the exchangeable protons (Gly(1,2,3,)) are largely exposed to solvent in the x-ray structure while the remaining three exchangeable protons of the ornithines are totally shielded from solvent. A very small number of normal mode displacements are found to expose the Orn(2) and Orn(3) amide groups while the Orn(1) amide proton remains shielded from solvent for all the paths studied. The effective paths for exposure of Orn(2) and Orn(3) correspond to the lowest frequency ( approximately 18 cm(-1)) motions. The paths are characterized in terms of the magnitude and energy of atomic displacements, correlated changes in dihedral angles, and the resulting changes in exposure and hydrogen bonding of exchangeable groups.

Journal Article↗
PubMed1982

Grant funding.

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National Institutes of Health (U.S.)↗
PubMed1982

Re-examination of rhodopsin structure by hydrogen exchange.

The hydrogen exchange behavior of rhodopsin was re-examined by studies of the protein in the disc membrane and after solubilization in octyl glucoside. The methods used measure either the peptide hydrogens alone (hydrogen-deuterium exchange by infrared spectroscopy) or all slowly exchanging hydrogens (hydrogen-tritium exchange by hel filtration). Under mild exchange conditions, disc membranes and solubilized lipid-free proteins show very similar exchange behavior, indicating the absence of slowly exchanging lipid protons. At high temperature, exchange of an additional large group of very slow peptide NH can be detected. The total number of slow hydrogens significantly exceeds the amide content, and apparently includes slowly exchanging protons from perhaps 40% of the protein's non-amide side chains. This is thought to require the involvement of many polar side chains in internal H-bonding. The exchange rates of the non-amide side chains sites have not been determined. However, to the extent that these contribute to the fast time region of the measured kinetic H-exchange curve, previously identified with exposed, non-H-bonded peptides, the estimate of freely exposed rhodopsin peptides must be reduced. The fraction of free peptides could range from a remarkably high value of 70% down to about 45%.

Animals↗
PubMed1981

Hydrogen-bonded structure of the complex N-linked fetuin glycopeptide.

The conformation of the N-linked complex glycopeptide of fetuin was examined with hydrogen-exchange techniques. The glycopeptide molecule contains eight acetamido hydrogens stemming from five N-acetylglucosamine residues and three N-acetylneuraminic acid residues and also one from the remaining sugar-peptide linkage. The hydrogen-exchange rates of these secondary amides were compared with small molecule model compounds having identical primary structures at their exchangeable hydrogen sites. Differences between the model rates and glycopeptide rates therefore cannot be accounted for by primary structure effects but reflect conformational features of the glycopeptide. Two glycopeptide hydrogens exhibit significantly hindered exchange; the rest exchange at the model rates. Removal of the three N-acetylneuraminic acid residues from terminal positions on the three branches of the glycopeptide removes the slowed hydrogens. The remaining ones continue to exchange at the model rate. These results indicate that two of the eight sugar acetamido hydrogens are involved in intramolecular hydrogen bonds. A likely structure includes two hydrogen bonds between the three N-acetylneuraminic acid residues. These two hydrogens, slowed to a moderate degree, reflect a preferred conformation stabilized by about 1 kcal/mol in free energy. The solution conformation of the glycopeptide suggested by these results is one that is partially ordered and can be easily modulated, owing to the relatively small amount of energy stabilizing the preferred conformation.

Carbohydrate Conformation↗
PubMed1980

Hydrogen--deuterium exchange analysis of ligand--macromolecule interactions: ethidium--deoxyribonucleic acid system.

The interaction between DNA and the intercalating dye, ethidium bromide, was studied by use of a novel approach in which hydrogen--deuterium (H--D) exchange between ethidium amino groups and solvent was measured spectrophotometrically in a stopped-flow mode. The method depends on the fact that ethidium H exchange is greatly slowed on complexation with DNA and that H--D exchange kinetics of the chromophore can be monitored via an accompanying change in its spectral absorbance. The H--D exchange dependent spectral character of ethidium was characterized, and the catalyzed exchange behavior of the free dye and the dye--DNA complex was studied. From such measurements, one can obtain rate and equilibrium constants for the interaction and possibly also some stereochemical information. The constants obtained were checked in more conventional mixing experiments. At 20 degrees C in high salt, the equilibrium binding constant is approximately 5 x 10(4) M-1, and on and off rate constants are 1.6 x 10(6) M-1 s-1 and 30 s-1, respectively. The results independently confirm that one dye molecule is bound for each 2--2.5 base pairs. The method should be applicable to a range of binding interactions. Among other advantages, this approach can allow tight binding interactions to be studied at concentrations of the reactants far above the characteristic Kdiss value.

Animals↗
PubMed1980

A high energy structure change in hemoglobin studied by difference hydrogen exchange.

The hydrogen exchange behavior of a small allosterically responsive set of exchanging hydrogens was studied in hemoglobin A and in some chemically modified hemoglobins. The set experiences an exceptionally large change in exchange rate through hemoglobin's allosteric transition. This indicates, according to the local unfolding model of H-exchange, that a large change in allosteric free energy impinges on the opening segment that exposes these protons to exchange. In oxyhemoglobin the set consists of 5 to 6 protons which exchange with a half-time of 20 s at pH 7.4 and 0 degrees C. In deoxyhemoglobin the set splits into a slower and a faster half. The slower 3 protons exchange more slowly than in oxyhemoglobin by a factor of 5000 (26 h half-time) and are 5-fold slower still in the presence of pyrophosphate or inositol hexaphosphate (136 h half-time). The other 2 to 3 protons exchange about 20-fold faster in both cases (about 2 h and 10 h half-times). The effect of some chemical modifications was tested, including reaction with iodoacetamide and N-ethylmaleimide and cleavage with carboxypeptidases A and B. In all cases the 3 slower protons continue to behave as a cohesive set and in the various modified deoxyhemoglobins their exchange is accelerated by factors ranging between 1 and 3 decades. These factors correlate with the effect of the different modifications on hemoglobin cooperativity.

Acetamides↗
PubMed1980

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach.

Deuterium↗
PubMed1980

Nature of the open state in long polynucleotide double helices: possibility of soliton excitations.

The existence of transiently open states in DNA and synthetic polynucleotide double helices has been demonstrated by hydrogen exchange measurements; base pairs reversibly separate and reclose, exposing nucleotide protons to exchange with solvent protons. Recently it has been possible to define the equilibrium, kinetic, and activation parameters of the major open state that determines base pair hydrogen exchange. However, there is no direct information at the moment about the conformation of the open form. Here we consider the possibility that the low energy and slow opening and closing rates observed reflect a deformation involving several adjacent base pairs. Assuming a mobile open unit capable of diffusing along the double helix, we find that available data are consistent with structures of 10 or so adjacent open pairs. It is further suggested that these structures correspond to thermally induced soliton excitations of the double helix, which retain coherence by sharing the energy of a twist deformation among several base pairs. Solitons are nonlinear excitations that can travel as coherent solitary waves, and have been recognized as an important mechanism for mediating conformational changes in polymers and condensed systems generally. Comparison of the double helix with simple mechanical analogs suggests that soliton excitations may well exist within DNA chains, and the present analysis shows that the hydrogen exchange open state is consistent with these.

Base Sequence↗
PubMed1978

Sickle hemoglobin gelation. Reaction order and critical nucleus size.

Sickle hemoglobin (Hb S) gelation displays kinetics consistent with a rate-limiting nucleation step. The approximate size of the critical nucleus can be inferred from the order of the reaction with respect to Hb S activity, but determination of the reaction order is complicated by the fact that Hb S activity is substantially different from Hb S concentration at the high protein concentrations required for gelation. Equilibrium and kinetic experiments on Hb S gelation were designed to evaluate the relative activity coefficient of Hb S as a function of concentration. These experiments used non-Hb S proteins to mimic, and thus evaluate, the effect on activity coefficients of increasing Hb S concentration. At Hb S concentrations near 20% the change in Hb S activity coefficient generates two-thirds of the apparent dependence of nucleation rate on Hb S concentration. When this effect is explicitly accounted for, the nucleation reaction is seen to be approximately 10th-order with respect to effective number concentration of Hb S. The closeness of the reaction order to the number of strands in models of Hb S fibers suggests a nucleus close to the size of one turn of the Hb S fiber. These experiments introduce a new approach to the study of Hb S gelation, the equal activity isotherm, used here also to show that Hb S.Hb A (normal adult hemoglobin) hybrids do incorporate into growing nuclei and stable microtubules but that A.S hybridization is neutral with respect to promotion of Hb S nucleation and the sol-gel equilibrium.

Chemical Phenomena↗
PubMed1977

Hydrogen exchange study of membrane-bound rhodopsin. I. Protein structure.

Structural parameters of rhodopsin in disc membrane preparations from frog and cattle were studied by hydrogen exchange methods. The method measures the exchange of protein amide hydrogens with water and can distinguish protons which are internally bonded from those which are hydrogen-bonded to water. The results show that about 70% of rhodopsin's peptide group protons are exposed to water. The identification of these groups as free peptides was made initially on the usual basis of the identity of their exchange rate with the well characterized free peptide rate; other experiments specifically excluded contributions from lipids, protein side chains, adventitious mucopolysaccharides, and intradisc water. In contrast to rhodopsin, other proteins generally have only 20 to 40% free peptide groups. Apparently rhodopsin has some unusual structural feature. Our results together with available information on rhodopsin suggest that a considerable length of its polypeptide chain is arranged at the surface of a channel of water penetrating into the membrane. Physicochemical considerations indicate that such a channel would have to be quite wide, 10 to 12 A or more, to explain the hydrogen exchange results.

Animals↗
PubMed1977

Hydrogen exchange study of membrane-bound rhodopsin. II. Light-induced protein structure change.

Hydrogen exchange studies of rhodopsin in disc membranes demonstrated that photolysis induces changes in the protein itself. Two different altered forms were detected. A late photointermediate in the bleaching sequence, which can be identified with metarhodopsin II, displays accelerated exchange. Subsequently, at the stage of fully bleached opsin, exchange becomes even slower than in rhodopsin. These changes involve only a small fraction of the protein's internally hydrogen-bonded peptide groups. The unusually large fraction of exposed peptide hydrogens observed previously for rhodopsin is unaltered in the photolyzed forms.

Animals↗