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Dive into the research topics where S.W. Englander is active.

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Featured researches published by S.W. Englander.


Quarterly Reviews of Biophysics | 1983

Hydrogen exchange and structural dynamics of proteins and nucleic acids.

S.W. Englander; Neville R. Kallenbach

Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid. Most structural work to date has centred upon the native state of macromolecules, the most probable macromolecular form. But the native state of a molecule is merely its most abundant form, certainly not its only form. Thermodynamics requires that all other possible structural forms, however improbable, must also exist, albeit with representation corresponding to the factor exp( — G i / RT ) for each state of free energy G i (see Moelwyn-Hughes, 1961), and one appreciates that each molecule within a population of molecules will in time explore the vast ensemble of possible structural states.


Analytical Biochemistry | 1987

Biochemistry without oxygen

S.W. Englander; Calhoun Db; Joan J. Englander

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.


Archives of Biochemistry and Biophysics | 1957

Optical methods for measuring nucleoprotein and nucleic acid concentrations

S.W. Englander; Herman T. Epstein

A discussion of the use of a differential refractometer to measure nucleoprotein concentration and of the Beckman spectrophotometer to measure nucleic acid concentration suggests that the measurements may be made with an accuracy and sensitivity comparable to those of the standard biochemical methods. Nucleoprotein concentrations are obtained by dividing the refractive-index increment by 0.00180. The optical density at 260 mμ, corrected for scattering and for protein absorption, is divided by 25.3 sq. cm./mg. to give RNA concentration at pH 5.7, and is divided by 20 sq. cm./mg. to give DNA concentration at pH 7.0. These methods are rapid and do not involve destruction of samples. The methods were applied to solutions of coliphage T2 and plant viruses TMV and SBMV. The nucleic acid contents were, respectively, 50, 5.6, and 21 %, all in good agreement with values in the literature.


Biophysical Journal | 1980

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

S.W. Englander; Calhoun Db; Joan J. Englander; Neville R. Kallenbach; R.K.H. Liem; E.L. Malin; C. Mandal; J.R. Rogero

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.


Journal of Molecular Biology | 1972

Hydrogen exchange study of some polynucleotides and transfer RNA.

Joan J. Englander; Neville R. Kallenbach; S.W. Englander

Abstract The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC). Studies With the synthetic polynucleotides show that, to interpret nucleic acid hydrogen exchange data in terms of quantity of base-paired structure, one must count 5 H for each G · C pair and 2 or 3 for A · U. Both poly(rG) · poly(rC) and poly(dG) · poly(dC) clearly show 5 slowly exchanging H per base pair. For A · U and I · C only 2 were detected, though other workers have found 3 for some A-T systems. These are all base-pair-bound H. The ribose OH is too fast to measure. The reasons for the surprisingly slow exchange of the exposed NH2 protons are unknown. The hydrogen exchange-rate behavior found for the polynucleotides suggests that some local structural distortion is necessary for any of the exchangeable H to react, including the exposed NH2 protons, and that the distortion important for hydrogen exchange is different from that occurring in thermal denaturation. All the tRNA samples show very similar hydrogen exchange profiles. The pure samples (formylmethionine and tyrosine tRNA from Escherichia coli) have ~120 slowly exchanging protons, far more than the ~55 Watson-Crick hydrogen bonds in the simple cloverleaf models. With the above numeration, however, the cloverleaf models for the two pure tRNA samples account for all but approx-imately 20 of their slowly exchanging H. The excess of 20 H is very close to the number required by models having extra tertiary structure. The tRNAs were found to exchange more slowly even than poly(rG) · poly(rC) and to have a unique salt and pH dependence. These anomalies could also be explained by the presence of some tertiary folding. Unacylated and 70% aminoacylated E. coli tRNAfMet were found to have identical hydrogen exchange behavior, suggesting absence of structure change upon aminoacylation. A method was developed for isolating authentic poly(rG) from normally heterodisperse mixtures by Sephadex gel filtration in 90% dimethyl sulfoxide. Formation of the 1:1 poly(rG) · poly(rC) complex was achieved by mixing-experiments in concentrated urea solutions at high temperature.


Annals of the New York Academy of Sciences | 1975

MEASUREMENT OF STRUCTURAL AND FREE ENERGY CHANGES IN HEMOGLOBIN BY HYDROGEN EXCHANGE METHODS

S.W. Englander

Porphyrins are very generally found in the company of proteins, and a lot of effort has been devoted t o finding out how they interact. Many studies have focused on three-dimensional structure and change in three-dimensional structure. Such studies have supplied important parts of the picture; but also fundamental in these interactions are the energy relationships, and about these we know very little. For example, consider the changes in protein structure that occur when the heme iron of cytochrome c is oxidized and reduced. According t o the theory of linked functions, the generation of these structure changes requires a reciprocal change in the redox potential of the complex, a change which depends, however, not on the geometric size or quality of the structure changes but on their free energy. Similarly, when oxygen is bound t o the heme iron of hemoglobin, protein structure changes are generated at the exact expense of oxygen-heme binding energy, and most interestingly, some of the binding energy is transmitted, in the form of energetic structure changes, to distant binding sites where it reappears as binding energy. The geometry of structure change will determine the pathways of energy flow and action, but largeappearing changes may have little free energy while sub-hgstrom distortions may carry large energies. Thus, as has long been appreciated, the qualitative information now available on three-dimensional structure in these systems is essential for the understanding of these processes, but as Lord Kelvin stressed, qualitative information alone is not enough. The functional impact of any structure change, its contribution t o allosteric signal transmission and to modification of protein function, can only be ascertained by measuring quantitatively its free energy content. We have been studying structure change in hemoglobin by use of a new hydrogen exchange method. It now appears that with this method we can detect individually the different changes in three-dimensional structure that occur within the protein. Beyond this, the method is sensitive t o what might be called


Journal of Molecular Biology | 1969

Measurement of the free and the H-bonded amides of myoglobin☆

S.W. Englander; R. Staley

Abstract It has become abundantly clear that the rather slowly exchanging hydrogens of a protein do not simply relate to α-helix content. Some of the other inferences reached in the early period of protein hydrogen exchange work do still appear to be valid, namely, that most side chain hydrogens exchange “instantaneously” and that H-bonded amides exchange very slowly. However, in conflict with the older equation of slow hydrogens with α-helix are the more recently demonstrated facts that proteins possess H-bonded amides other than those in α-helix, which may also be expected to exchange very slowly, and that amides even when free can exchange measurably slowly, though perhaps not nearly as slowly as those in hydrogen bonds. Thus, rather than absolute separation between “instantaneous” and slow hydrogen exchange classes, differentiation between relatively faster and relatively slower kinetic classes seems indicated. Such a separation might provide a measure of structure representing the over-all number of H-bonded amides rather than just α-helix. A successful test of this expectation was carried out with myoglobin. To perform this test, the fairly rapidly exchanging hydrogens of myoglobin were preferentially labeled by brief exposure to tritiated water, and their exchange-out behavior was studied. The exchange-out of these faster hydrogens convincingly match theoretical exchange curves for free amide groups. These theoretical curves were constructed using the numbers of free amide groups in the myoglobin crystallographic model and hydrogen exchange rates found for such groups in model polymers. By a less demanding variant of this same exchange-in/exchange-out approach, the number of abnormally slowly exchanging hydrogens was independently determined and found to match the number of internally H-bonded amides in the myoglobin model structure. The good agreement found is taken as establishing the identity of the free and the H-bonded amides of myoglobin with distinguishable kinetic classes of exchanging hydrogens. The results demonstrate that, for this protein, free amides show simple and predictable hydrogen exchange behavior, that essentially all H-bonded amides exchange much more slowly, and that essentially all the other side chains exchange considerably faster. Evidence was found for the importance of an “opening”-dependent pathway for slowly exchanging, H-bonded hydrogens of myoglobin, as opposed to some direct exchange mechanism not dependent on conformational movement. The present study also provides another parameter common to both crystalline and dissolved myoglobin, namely, the number of H-bonded (donor) peptide groups.


Biophysical Journal | 1990

Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c

Yiqing Feng; Heinrich Roder; S.W. Englander

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.


Biophysical Journal | 1978

Sickle hemoglobin gelation. Reaction order and critical nucleus size

M.J. Behe; S.W. Englander

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.


Nature | 1975

Molecular structure of membrane-bound rhodopsin

N W Downer; S.W. Englander

WE have results which suggest a structure for the membrane-bound sensory protein, rhodopsin. This structure has novel features which are of considerable biological interest.

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Calhoun Db

University of Pennsylvania

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Joan J. Englander

University of Pennsylvania

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Leland Mayne

University of Pennsylvania

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A.J. Wand

University of Pennsylvania

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C. Mandal

University of Pennsylvania

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Yiqing Feng

University of Pennsylvania

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