Hydrogen Exchange Methods Research Articles

Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study.

Medium-resolution hydrogen exchange methods have been used to examine the solvent accessibility of seven peptides in the regulatory subunit (r2) of Escherichia coli aspartate transcarbamylase in the presence and absence of ATP and CTP. Both nucleotides are allosteric effectors of the holoenzyme; binding of ATP increases the affinity of the holoenzyme for the substrate L-Asp, while CTP has the opposite effect. Following Rosa and Richards (1979, 1981, 1982) and Englander et al. (1983, 1985), exchange-out curves for individual peptides were generated by adjusting the pH to 2.7 to quench exchange-out, digesting the protein with pepsin, separating peptides by reverse-phase HPLC, determining their radioactivity, and correcting for radioactivity lost during the analysis. Sixteen peptides from segments 1-11 and 76-153 were identified by amino acid and N-terminal analysis. Nine fell in regions where background was too high or were present at too low concentrations for exchange to be monitored. The number of protons whose exchange could be followed in peptides 1-11, 76-91, 78-90, 84-101, 93-112, 108-114, and 115-125 ranged from approximately 1 (1-11, 108-114) to 10 (84-101) and 11 (93-112). The pattern of results obtained suggests that the structure of r2 in solution is similar to that of the regulatory subunits in crystalline ATCase. Both CTP and ATP reduce rates of exchange from all seven peptides except 115-125. Although CTP slows exchange more than ATP, the effect is small except for peptides 76-91 and 78-90 which are near the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the “closed” macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.

22]Individual breathing reactions measured by functional labeling and hydrogen exchange methods

22]Individual breathing reactions measured by functional labeling and hydrogen exchange methods

Comparative study of the conformational transitions of frog and rabbit phosphorylases B

1. 1. Conformational motility of the purified muscle glycogen phosphorylase B from two species of vertebrates (rabbit and frog) was investigated by the Hydrogen-Exchange method and Infrared Spectrometry. 2. 2. The experimental results of the 1H- 2H exchange were expressed interms of the probability of P of exposure to isotopic solvent of phosphorylase peptide groups and in terms of the corresponding changes in standard free energy ΔG 0. 3. 3. The combined methods used didn't show considerable differences of the protein conformations in the physiological pH region but rabbit phosphorylase was only characterized by rather more compact structure in comparison with frog phosphorylase.

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature, pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding. The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C. S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10 3-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.

Structure dynamics of proteins by hydrogen exchange methods

Hydrogen exchange data can monitor the kinetic behavior of all but the most rapidly exchanging protons. Our experiments with lysozyme showed that the rate constants could be characterized by the function, where H(t) = hydrogens remaining per molecule at time t, and b = maximum number of observable hydrogens. The power law term, (1 + at)/sup -n/, describes exchange from the native state of the protein, while the term exp(-ct) accounts for exchange from the thermably unfolded state. Eq. 1 can be transformed to yield a distribution function, which describes the probability of observing H(t) governed by rate constants over the interval of logk to logk + dlogk. G(k) is bimodal with the asymptotic behavior attributable to thermal unfolding. In this communication we shall show the utility of such a distribution function.

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.

Hydrogen-deuterium exchange studies of proteins and nucleic acids

A kinetic study of the hydrogen exchange reaction of a protein or a nucleic acid can provide useful information on the structure and fluctuation of such a biological macromolecule. However, reactions faster than 10s are not easily traced by the methods used in earlier studies. Recent introduction of stopped-flow ultraviolet absorption and emission spectrophotometry into the hydrogen exchange studies of proteins and nucleic acids promises great development in this field. By this new technique we can now explore the millisecond region of the time-scale of hydrogen exchange kinetics. In this discussion we demonstrate some new aspects of the dynamic properties of proteins and nucleic acids on the basis of our results obtained by the stopped flow hydrogen exchange method.

A simple hydrogen exchange method for cross-linked protein crystals

A method is presented for measurement of hydrogen-tritium exchange in insoluble protein samples using a specially constructed closed chamber type filtration device. The protein sample is initially exhaustively tritiated at high pH, and back-exchange is initiated in the filtration unit by removal of tritiated solvent from the sample by rinsing with buffer of desired pH. The back-exchange is followed by sequential samplings of the tritium liberated to the solvent and it is completed by increasing pH. Experiments with monoclinic hen egg white lysozyme crystals, insolubilized by cross-linking with glutaraldehyde, could be followed as early as 20 seconds from initiation of back-exchange and exchange curves were highly reproducible. Exchange curves were identical with those of the dissolved enzyme at pH 3 and only small differences were detected between the two states at pH 6.8, supporting the idea of negligible change of protein motility upon crystallization and cross-linking. Diffusional equilibration processes are discussed.

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.

Hydrogen Exchange Studies of Respiratory Proteins: IV. A NEW, LIGAND-RESPONSIVE CLASS IN HEMOGLOBIN

Abstract As part of an ongoing survey, the early time region of hemoglobin's hydrogen exchange curve was studied to find hydrogens that respond to allosteric structure change. A difference hydrogen exchange method, previously designed for this kind of study, was used. Among the approximately 25 hydrogens per subunit studied, 7 respond to ligand binding by accelerating 14-fold in exchange rate; the remainder are ligand-indifferent. The responsive hydrogens form a first order kinetic class in both liganded and deoxyhemoglobin, and this gives further support to the breathing picture of hydrogen exchange. The increase in exchange rate observed indicates that the segment holding these hydrogens experiences a net structural free energy change worth +1.5 Cal in the allosteric transition.

A study by the hydrogen-exchange method of the complex formed between the basic pancreatic trypsin inhibitor and trypsin.

The kinetics of the 1H–2H exchange of the peptide groups is studied in 2H2O solutions of the basic pancreatic trypsin inhibitor, of trypsin, and of the inhibitor-trypsin complex. In average the exchange rates in solutions of the complex are slower than the exchange rates in solutions of the constituent proteins. The experimental results are expressed in terms of the probability ϱ of exposure to the solvent of the peptide groups of the proteins, and in the changes in standard free energy ΔG° of the conformational transitions by which the exposure is effected. The distribution of values of these parameters on the peptide groups of the proteins is discussed.

Native collagen has a two-bonded structure

A number of structural models that differ sterically and in interpeptide hydrogen-bonding pattern have been put forward for the native collagen molecule. These have proven remarkably difficult to distinguish by X-ray diffraction and other methods. The various models have generally been divided into a “one-bonded” class (one hydrogen bond per triad of amino acid residues) and a “two-bonded” class (nearly two bonds per triad). To discriminate between the models, we have applied hydrogen-tritium exchange methods to native soluble ichthyocol (carp swim bladder) collagen. Free and hydrogen-bonded peptide groups were recognized by their hydrogen exchange rates. Under two different solution conditions, a set of peptide hydrogens was found to display exchange rates nearly identical to those previously calibrated for free peptides in model systems and, therefore, was taken to represent free peptides. The remaining hydrogens exchanged much more slowly and were assigned to internally hydrogen-bonded peptide groups. The numbers of free and of internally bonded peptide hydrogens were measured independently in different kinds of exchange experiments. They coincide with the numeration required for two-bonded models. The results appear to exclude one-bonded models for ichthyocol collagen but cannot distinguish between the earlier two-bonded model, which contains two direct peptide to peptide hydrogen bonds per triad, and the more recent proposal involving one direct bond and one cross-bridging water molecule. The results, together with other data in the literature, indicate that native collagens from other species may also exist in a two-bonded structure, though some synthetic sequence polypeptides related to collagen do clearly assume a one-bonded structure. The results are relevant also to the general problem of structure analysis by hydrogen exchange methods. It has previously been demonstrated that free peptide hydrogens exchange at precisely predictable rates in small molecules, in oligo- and polypeptides, and in a globular protein. The present data extend this conclusion to a fibrous protein. Thus the techniques used here may be quite generally applied to distinguish and to count free and internally bonded peptide groups in protein and polypeptide systems.

Hydrogen Exchange Studies of Respiratory Proteins: III. STRUCTURAL AND FREE ENERGY CHANGES IN HEMOGLOBIN BY USE OF A DIFFERENCE METHOD

Abstract A difference hydrogen exchange method was developed and applied to hemoglobin. The method can be used to pick out, from the total hydrogen exchange curve of a protein, the hydrogen exchange behavior of just those segments of the protein that alter in exchange rate in response to structural change. With the method even quite subtle structure changes can be individually recognized since protein mechanisms act to translate structure changes into greatly amplified hydrogen exchange signals. A fraction of hemoglobin's hydrogen exchange curve containing about 40% of the protein's hydrogen-bonded peptide groups was analyzed. Among these the difference techniques portrayed the exchange behavior of 20 ligand-responsive hydrogens in reduced and liganded hemoglobin and also the exchange of 30 ligand-indifferent hydrogens. The ligand-responsive hydrogens fall into three kinetically distinct groups having 3, 8, and 9 hydrogens on a per subunit basis. All three groups experience much faster exchange in the liganded form of the protein. The exchange character of these groups demonstrates that their exchange, and probably protein hydrogen exchange in general, is controlled by breathing reactions—cooperative, reversible denaturations—of the segments of protein structure holding them. This picture leads immediately to a quantitative relationship connecting energetic structure change, breathing change, and alteration in hydrogen exchange rate. The results, interpreted in this way, show that when ligand is bound to hemoglobin the two larger responsive segments studied are destabilized by close to 2 Cal in structural free energy and the smaller one by perhaps 4 Cal.

The accessibility of hydrocelluloses and oligosaccharides by hydrogen exchange

AbstractThe accessibility of hydrocelluloses and oligosaccharides was measured by hydrogen exchange methods. It is shown that the accessibility of various celluloses remains constant during prolonged heterogeneous hydrolysis. This is at variance with the fringed micellar concept of the fine structure of cellulose, on the basis of which a substantial decrease in accessibility would be expected during hydrolysis.Accessibility measurements on amorphous and crystalline oligosaccharides suggest that the accessibility may be related to the perfection of the crystal lattice and does not measure the crystalline‐amorphous ratio as previously supposed.Finally, the results are discussed in relation to a recent theory of the fine structure of cellulose.

A convenient method for the preparation of S-peptide from bovine pancreatic ribonuclease

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥103-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.

HYDROGEN EXCHANGE STUDIES OF DNA STRUCTURE.

DNA unwinding, and may well control the accessibility of the structure to nucleases and polymerases. In principle, hydrogen exchange methods afford the most direct experimental approach to the dynamic properties of the DNA helix. Hydrogen exchange has frequently been used to study the conformations of proteins. The results of such measurements have been interpreted on the basis that a potentially exchangeable hydrogen atom which is involved in intramolecular hydrogen bonding (and/or buried in the interior of a macromolecule) exchanges much more slowly with solvent hydrogen than one not so constrained. With most proteins conditions of exchange can be found for which rates of exchange are measured in hours, or even days. By analogy, the potential applicability of hydrogen exchange methods to a study of hydrogen-bonding and conformation in DNA has been recognized for some time. However, Bradbury et alt. found that essentially complete hydrogen exchange occurred during the 3 min required for sample manipulation following the transfer of a fully deuterated DNA film from a D20 to an H20 environment. As a result of this experience and others (unpublished), it has been generally accepted that the hydrogens of DNA exchange instantaneously and that the methods usually applied to proteins cannot be used. Recently, Englander' has developed a new experimental approach to hydrogen exchange. In this method the macromolecule of interest is incubated to equilibrium with tritiated water, and then quickly separated from unbound tritiumn by gel filtration. The bound tritium is then measured by liquid scintillation counting. This technique has several advantages over the nmore conventional hydrogen exchange methods: it is a true tracer method which does not involve the massive substitution with deuterium characteristic of the older methods, no drying steps are involved so that the macromolecule is kept under controlled environmental conditions at all times, and the method is quite fast, complete separation of the labeled macromolecule from free background tritium being attained in a little over I min. In this paper we demonstrate that under appropriate conditions the exchange of structural hydrogens in DNA (i.e., those involved in interchain hydrogen bonding) can be measured with considerable precision using the tritium-gel filtration tech363

Tritiated digoxin studies in human subjects.

A number of publications have appeared in recent years regarding the metabolic behavior, fate, and excretion of C14-labeled digitoxin.1-8Labeled digoxin has received little attention.9-11The preparation of a C14-labeled compound has not been practical because of the expense and uncertainty of the digoxin content of digitalis glycosides. The possibility of a tritium (H3) label was explored and was found to be both cheap and practical utilizing the Wilzbach hydrogen exchange method. The present study was undertaken to study the serum levels, absorption, and excretion of tritium-labeled digoxin*in human subjects after singledose oral digitalization. Most of the metabolic studies of unlabeled digoxin12,13have revealed the presence of unchanged digoxin and metabolite B in the urine of both rats and humans. Brown and Wright14reported the presence of metabolite F in the tissue of rats. Metabolite B has not been demonstrated