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Biomedical subjects

S W Englander

Publications and source records attributed to S W Englander.

At least 73 records · Page 4Linked to original sources
PubMed1987

Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins.

A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C alpha H-C beta H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequence. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposes an internally confirmatory property on each forward step.

Algorithms↗
PubMed1987

On the prevalence of room-temperature protein phosphorescence.

A large number of proteins were tested for the property of intrinsic phosphorescence in deoxygenated aqueous solution at room temperature. The majority of proteins exhibit phosphorescence under normal solution conditions. Phosphorescence lifetimes from 0.5 millisecond to 2 seconds were observed in three-fourths of the proteins tested. The lifetime appears to correlate with relative isolation of the tryptophan indole side chain from solvent. With few exceptions, proteins in general can be expected to display a phosphorescence lifetime greater than 30 microseconds. This widespread characteristic of proteins has been largely overlooked because long-lived phosphorescence is highly sensitive to quenching by low levels of dissolved oxygen in solution. Protein phosphorescence offers a new time domain and a far wider dynamic range than has been used before for photoluminescence experimentation.

Fluorescence↗
PubMed1987

Hydrogen-tritium exchange survey of allosteric effects in hemoglobin.

The oxy and deoxy forms of hemoglobin display major differences in H-exchange behavior. Hydrogen-tritium exchange experiments on hemoglobin were performed in the low-resolution mode to observe the dependence of these differences on pH (Bohr effect), organic phosphates, and salt. Unlike a prior report, increasing pH was found to decrease the oxy-deoxy difference monotonically, in general accordance with the alkaline Bohr effect. A prior report that the H-exchange difference between oxy- and deoxyhemoglobin vanishes at pH 9, and thus appears to reflect the Bohr effect alone, was found to be due to the borate buffer used, which at high pH tends to abolish the oxy-deoxy difference in a limited region of the H-exchange curve. Effects on hemoglobin H exchange due to organic phosphates parallel the differential binding of these agents (inositol hexaphosphate more than diphosphoglycerate, deoxy more than oxy, at low pH more than at high pH). Added salt slows H exchange of deoxyhemoglobin and has no effect on the oxy form. These results display the sensitivity of simple H-exchange measurements for finding and characterizing effects on structure and dynamics that may occur anywhere in the protein and help to define conditions for higher resolution approaches that can localize the changes observed.

Allosteric Regulation↗
PubMed1987

Biochemistry without oxygen.

Published procedures for experimentation under anoxic conditions generally involve specialized apparatus that hinders the easy manipulation of experimental samples. We describe here some procedures that rapidly remove oxygen from experimental solutions, maintain anoxia with simple equipment for long periods of time, and do not interfere with normal sample addition and removal, spectrometric measurements, chromatographic manipulations, and the like. Anoxia can be achieved and maintained by the use of an enzyme system (glucose oxidase, glucose, catalase), or an inorganic oxygen-reducing system (ferrous pyrophosphate), or dithionite. Physical isolation of experimental samples from atmospheric oxygen can be maintained by continuous flushing with treated argon gas and/or by an overlay of heavy mineral oil.

Argon↗
PubMed1986

Allosteric sensitivity in hemoglobin at the alpha-subunit N-terminus studied by hydrogen exchange.

Allosteric structure change in human hemoglobin was studied by hydrogen-tritium-exchange methods. The functional labeling method used takes advantage of the change in H-exchange rate at allosterically involved sites to selectively label, with tritium, H-exchange sites that are fast in one protein state and slow in another. The position of the labeled sites can then be located by the medium-resolution fragmentation-separation method. These methods reveal 5 allosterically sensitive, H-bonded, peptide NH's within the first 12 residues of the alpha chain. All five exchange with solvent protons at similar rates in deoxyhemoglobin (T form), and all shift to a new rate, about 30-fold faster, in the liganded protein (R) form. This indicates a decrease in structural stability at the alpha-chain N-terminus in going from the T to the R form, consistent with the loss of stabilizing interactions in that segment. The results indicate a loss of perhaps 2 kcal/mol in stabilization free energy and thus document a significant role for changes at the alpha-chain N-terminus in the allosteric transition.

Allosteric Regulation↗
PubMed1986

Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix.

The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolve many ambiguities in intraresidue J-coupled connectivities and interresidue NOE connectivities. This required no explicit knowledge of the solution structure. The pattern of NOEs found is consistent with a regular alpha helix between glycine-6 and lysine-13; H bonding continues at least through alanine-15 [see Wand, A.J., Roder, H., & Englander, S. W. (1986) Biochemistry (following paper in this issue)]. Chain disorder occurs at the N-terminus. There is no indication of significant spin diffusion among the backbone amide and alpha-protons of this 12.4-kilodalton protein even at the longest NOE mixing time used (140 ms).

Amino Acid Sequence↗
PubMed1986

Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix.

The hydrogen exchange behavior of the N-terminal helical segment in horse heart cytochrome c was studied in both the reduced and the oxidized forms by use of two-dimensional nuclear magnetic resonance methods. The amide protons of the first six residues are not H bonded and exchange rapidly with solvent protons. The most N-terminal H-bonded groups--the amide NH of Lys-7 to Phe-10--exhibit a sharp gradient in exchange rate indicative of dynamic fraying behavior, consistent with statistical-mechanical principles. This occurs identically in both reduced and oxidized cytochrome c. In the oxidized form, residues 11-14, which form the last helical turn, all exchange with a similar rate, about one million times slower than the rate characteristic of freely exposed peptide NH, even though some are on the aqueous face of the helix and others are fully buried. These and similar observations in several other proteins appear to document local cooperative unfolding reactions as determinants of protein H exchange reactions. The N-terminal segment of cytochrome c is insensitive to the heme redox state, as in the crystallographic model, except for residues closest to the heme (Cys-14 and Ala-15), which exchange about 15-fold more slowly in the reduced form. The cytochrome c H exchange results can be further considered in terms of the conformation of the native and the transiently unfolded forms and their free energy relationships in both the reduced and the oxidized states.

Animals↗
PubMed1986

Protein fluorescence quenching by small molecules: protein penetration versus solvent exposure.

Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pattern of fluorescence quenching exhibited by quenching molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves protein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quenching, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For several proteins having tryptophans known to be buried, no quenching occurs. These results are not consistent with the view that the kinds of small molecules studied can quite generally penetrate into and diffuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion.

Alcohol Dehydrogenase↗
PubMed1985

Two-dimensional 1H NMR studies of cytochrome c.

Two-dimensional nuclear magnetic resonance techniques were used to assign the NH, C alpha H, and C beta H protons of over 60 of the 104 amino acid residues in the 1H NMR spectrum of horse ferrocytochrome c. The majority of these amino acids were completely assigned. Assignments were based on the analysis of two-dimensional J-correlated (COSY), nuclear Overhauser effect (NOESY), and relayed COSY spectra and on comparisons of the J-correlated spectra of various cytochrome c species. Spin diffusion is not a problem with monomeric proteins the size of cytochrome c. Here these advances are illustrated with data that lead to the assignment of the heme-associated residues cysteine-14 and tryptophan-59, the axial ligands methionine-80 and histidine-18, the entire N-terminal helix, and several other amino acid spin systems. With these approaches, structure, structure change, the internal dynamics of cytochrome c, and the interaction of these with function are being studied, especially by observation of the hydrogen exchange behavior of essentially all the H-bonded amides and some side chain protons in both the reduced and oxidized proteins.

Animals↗
PubMed1985

Protein hydrogen exchange studied by the fragment separation method.

The potential of hydrogen-exchange studies for providing detailed information on protein structure and structural dynamics has not yet been realized, largely because of the continuing inability to correlate measured exchange behavior with the parts of a protein that generate that behavior. J. Rosa and F. M. Richards (1979, J. Mol. Biol. 133, 399-416) pioneered a promising approach to this problem in which tritium label at exchangeable proton sites can be located by fragmenting the protein, separating the fragments, and measuring the label carried by each fragment. However, severe losses of tritium label during the fragment separation steps have so far rendered the results ambiguous. This paper describes methods that minimize losses of tritium label during the fragment separation steps and correct for losses that do occur so that the label can be unambiguously located and even quantified. Steps that promote adequate fragment isolation are also described.

Chemical Phenomena↗
PubMed1985

Internal protein motions, concentrated glycerol, and hydrogen exchange studied in myoglobin.

Experiments were carried out to measure the effect of concentrations of glycerol on H-exchange (HX) rates by using myoglobin as a test protein. Concentrated glycerol has only a small slowing effect on the HX kinetics of freely exposed amides, studied in a small molecule model (acetamide). Larger effects occur in structured proteins. The effect of solvent glycerol on different parts of the HX curve of myoglobin was studied by use of a selective "kinetic labeling" approach. Concentrated glycerol exerts an apparently reverse effect on protein H exchange; the faster exchanging "surface" protons are least affected, while the slower and slower amide NH is further slowed by larger and larger factors. These results seem inconsistent with solvent penetration models which generally visualize slower and slower protons as being placed, and undergoing exchange, farther and farther from the solvent-protein interface. On the other hand, the results are as expected for the local unfolding model for protein H exchange since concentrated glycerol is known to stabilize proteins against unfolding. In the local unfolding model, slower exchanging protons are released by way of higher energy and therefore generally larger, unfolding reactions. Larger unfoldings must be more inhibited by the glycerol effect.

Animals↗
PubMed1983

Identification of an allosterically sensitive unfolding unit in hemoglobin.

Hydrogen-exchange studies locate a set of seven allosterically sensitive amide NH protons side by side around two turns of the F-FG helical segment in the hemoglobin beta chain. Some of these protons are on the aqueous protein surface and some deeply inside, yet they all exchange with solvent protons at similar rates. Further, they move in unison to a new common rate when hemoglobin changes its allosteric form. These observations and analogous results for other proteins appear to be inconsistent with penetration-dependent models which relate H-exchange rate to solvent accessibility in the native state. Rather, these results point to sizeable fluctuational distortions that make small sets of protons more or less equally accessible in some transient H-exchange transition state, as visualized in the local unfolding model. The set of allosterically sensitive protons studied here exchanges 30-fold faster in liganded hemoglobin than in the deoxy form. In terms of the unfolding model, this means that the F-FG structure is relatively destabilized in oxyhemoglobin, so that the allosterically linked change in structural free energy at F-FG favors the deoxy state. The 30-fold change in H-exchange rate suggests a contribution to the allosteric free energy by this segment of 2 kcal (1 cal = 4.184 J). These experiments utilized a labeling technique, described earlier, that selectively places tritium on sites whose H-exchange rates are sensitive to the protein functional state, and used a method introduced by Rosa & Richards (1979,1981) to locate this label in the protein. The latter method, which rapidly separates protein fragments under conditions that can preserve exchangeable label, was here brought to a more quantitative level. Taken together, these techniques provide a "functional labeling" method capable of selectively labeling and identifying protein segments that participate in functional interactions.

Allosteric Site↗
PubMed1983

Penetration of dioxygen into proteins studied by quenching of phosphorescence and fluorescence.

Experiments were done to measure the ability of dioxygen to collisionally quench the phosphorescent and fluorescent tryptophans in alcohol dehydrogenase and alkaline phosphatase. In all cases, luminescence is quenched with rate constants close to 1 x 10(9) M-1 s-1. The rate of reaching the buried tryptophans is little affected by solvent viscosity due to added glycerol. Quenching by dioxygen is not due to a protein-opening reaction. It appears to be rate limited by internal protein diffusion rather than at the entry step. Dioxygen appears to enter the proteins directly, as in liquidlike diffusion, rather than through transiently forming channels that are only present a small fraction of the time. A high-pressure oxygen system is described that considerably facilitates fluorescence quenching experiments.

Alcohol Dehydrogenase↗