Joan J. Englander

Mechanisms and uses of hydrogen exchange.

Recent work has largely completed our understanding of the hydrogen-exchange chemistry of unstructured proteins and nucleic acids. Some of the high-energy structural fluctuations that determine the hydrogen-exchange behavior of native macromolecules have been explained; others remain elusive. A growing number of applications are exploiting hydrogen-exchange behavior to study difficult molecular systems and elicit otherwise inaccessible information on protein structure, dynamics and energetics.

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.

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.

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.

Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry.

Abstract An exceedingly simple and convenient method is described for measuring the hydrogen-deuterium exchange behavior of peptide bond-containing molecules by ultraviolet spectrophotometry. The exchange reaction is initiated by diluting a sample from H 2 O into D 2 O, or the reverse, and can be followed by an easily observable optical density change in the region of peptide absorbance. The method, unlike infrared and magnetic resonance approaches, requires only small amounts of material and, unlike the tritium-Sephadex method, is not restricted to the study of large molecules. Calibrations are provided for exchange rate as a function of pD and temperature and for the change in absorbance per mole peptide group. With this information, the exchange curve to be expected for any peptide group exposed to solvent can be predicted. Comparison with the measured data can then identify peptide-group hydrogen bonding and can also give a measure of the stability of the hydrogen-bonded structure.

[15] Thermodynamic parameters from hydrogen exchange measurements

Just as exchangeable hydrogens that are controlled by global unfolding can be used to measure thermodynamic parameters at a global level, hydrogens that are exposed to exchange by local unfolding reactions may be used to obtain locally resolved energy parameters. Results with the hemoglobin system demonstrate the ability of HX methods to locate functionally important changes in a protein and to measure the energetic contribution of each. These results offer the promise that HX measurements may be used to delineate, in terms of definable bonds and their energies and interactions, the network of interactions that Hb and other proteins use to produce their various functions.

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 exchange study of some polynucleotides and transfer RNA.

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.

[3] Structure and energy change in hemoglobin by hydrogen exchange labeling

Publisher Summary Hydrogen exchange-labeling methods can in principle show which parts of hemoglobin are actively involved in the allosteric process and which are not. The approach derives from the local unfolding model. The local unfolding model for protein hydrogen exchange connects the exchange rate with local structural free energy. Thus the measurement of changes in hydrogen exchange (HX) rate may locate allosterically important changes and delineate the handling of allosteric energy in quantitative free-energy terms. The methods described in the chapter have made it possible to locate some of the important interactions and to measure their free-energy contribution to the overall allosteric transition. Results have demonstrated a quantitative relationship between structural free energy measured locally by these methods and globally by other established methods that involve the analysis of ligand-binding curves and the measurement of subunit-dissociation equilibria.

Allosteric energy at the hemoglobin beta chain C terminus studied by hydrogen exchange.

When hemoglobin switches from the deoxy (T) to the liganded (R) form, several of its peptide group NH experience a great increase in their rate of exchange with water. Selective labeling and fragment isolation experiments identify some of the sensitive protons as three to four near-neighbor H-bonded peptide NH placed between Ala140 beta and the C-terminal His146 beta residue. These NH have differing solvent accessibilities, yet all exchange at about the same rate, and they maintain a common rate in the face of modifications that change their exchange rate over a 1000-fold range. This suggests that their exchange is mediated by a concerted transient unfolding reaction. The removal of allosterically important salt links at the distant alpha subunit N termini (des-Arg141 alpha hemoglobin) has little if any effect on the indicator NH at the beta C terminus. This demonstrates the restricted reach of the separate allosteric interactions in the T form as well as the localized nature of the H-exchange probe. Breakage of a salt link at the beta chain C terminus (His146 beta to Asp94 beta) by chemical modification (NES-Cys93 beta hemoglobin) speeds exchange of the indicator peptide NH in T-state hemoglobin by six-fold, which corresponds to an allosteric destabilization at the C-terminal segment of 1 kcal (pH 7.4, 0 degrees C), according to local unfolding theory. This is in quantitative agreement with energy values obtainable from other measurements. These NH exchange with an average halftime of five hours in deoxy hemoglobin and 15 seconds in oxy hemoglobin. According to the unfolding model for protein H-exchange, the 1200-fold increase in rate indicates a loss of 3.8 kcal in structural stabilization free energy at or near the C terminus of each beta chain in the T to R transition (pH 7.4, 0 degrees C, with 2,3-diphosphoglycerate). This result together with other available data places about 70% of hemoglobins total allosterically significant structural energy change at the beta chain C termini.