Individual breathing reactions measured in hemoglobin by hydrogen exchange methods

Englander, S. Walter; Calhoun, D. B.; Englander, Joan J.; Kallenbach, Neville R.; Liem, R.K.H.; Malin, Edyth L.; Mandal, C.; Rogero, J.R.

doi:10.1016/s0006-3495(80)84991-5

Cited by 92 publications

(64 citation statements)

References 23 publications

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“…Selectivity and accuracy increase with the rate difference, i.e., with the importance of the structure change, and decrease to zero as the rate difference disappears. A fragment separation method can then identify the positions of the selectively labeled allosterically sensitive sites at medium resolution (6)(7)(8)(9). Samples taken after different exchange-out times are plunged into slow HX conditions (pH Ϸ2.5, 0°C) and quickly fragmented with acid protease.…”

Section: Methodsmentioning

confidence: 99%

“…Earlier work (5,6) developed a ''functional-labeling'' approach that can selectively label, by hydrogen-tritium (H-T) or hydrogen-deuterium (H-D) exchange, just those sites that change in any functional process. In favorable cases, the label can then be located at medium resolution by a proteolytic fragmentation method in which the fragments are quickly produced and then separated by HPLC under conditions where the loss of isotopic label is slow (6)(7)(8)(9).…”

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confidence: 99%

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

Englander

Mar

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

199

191

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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.H ydrogen exchange (HX) measurements can, in principle, locate protein-binding sites and structure changes and can quantify otherwise unavailable dynamic and energetic parameters (1-4). For relatively small proteins, HX can be measured at an amino acid resolved level by NMR methods. For larger, functionally more interesting proteins, other strategies are required. Earlier work (5, 6) developed a ''functional-labeling'' approach that can selectively label, by hydrogen-tritium (H-T) or hydrogen-deuterium (H-D) exchange, just those sites that change in any functional process. In favorable cases, the label can then be located at medium resolution by a proteolytic fragmentation method in which the fragments are quickly produced and then separated by HPLC under conditions where the loss of isotopic label is slow (6-9).To move toward higher resolution and more comprehensive coverage of target proteins, recent work in many laboratories has coupled the HPLC separation to a second dimension of fragment resolution by online MS (10, 11). These methods tend to be labor intensive and time consuming, with limitations in throughput and comprehensiveness and in the structural resolution of functionally important changes. This article merges previous HX functional labeling and fragment separation methods with an automated MS approach termed deuterium exchange MS (DXMS) (12-18).We are using Hb as a model system to study how protein molecules manage intramolecular signal transduction processes. Hb functions by transducing a part of the binding energy of its initially bound O 2 ligands into structure-change energy. The energy is carried through the protein to distant heme sites in the form of energetic structure changes, and there transduced back into binding energy. The initial reduced binding energy and the later enhanced binding produces the physiologically important sigmoid binding curve. In short, the currency of allosteric interactions is free energy. Trying to understand allostery without measuring free energy is like trying to understand an economic system without measuring money. A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Englander

Mar

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

199

191

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“…Table.2). The highly selective nature of hydrogen bond cleavage observed for these exchange paths is in contrast to proposedimechanisms that involve unfolding for which cooperative breaking of adjacent hydrogen bonds is likely to be important (1,5).…”

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confidence: 74%

“…Given (1)(2)(3)(4). However, there is still much uncertainty concerning a molecular interpretation of the experimental results (5)(6)(7)(8)(9). Attention is currently focused on two models for hydrogen exchange.…”

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confidence: 99%

Normal mode paths for hydrogen exchange in the peptide ferrichrome

Sheridan

Levy

Englander

1983

Proc. Natl. Acad. Sci. U.S.A.

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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"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 Orn2 and Orn3 amide groups while the Orn' amide proton remains shielded from solvent for all the paths studied. The effective paths for exposure of Orn2 and Orn3 correspond to the lowest frequency (18 cm (1)(2)(3)(4). However, there is still much uncertainty concerning a molecular interpretation of the experimental results (5-9). Attention is currently focused on two models for hydrogen exchange. One emphasizes the need for cooperative hydrogen bond breakage, which is suggested to occur via individual concerted unfoldings of small molecular segments. Alternatively, penetration models focus on diffusion of hydrogen exchange catalysts into the protein, utilizing multiple small structural fluctuations. There are other possible exchange mechanisms. In this communication we investigate paths for hydrogen exchange in the cyclic hexapeptide ferrichrome. The paths, which are obtained from calculations of the vibrational normal modes of ferrichrome, are characterized in terms of the magnitude and energy of atomic displacements and correlated changes in dihedral angles and the resulting changes in exposure and hydrogen bonding of exchangeable groups.The cyclic hexapeptide [Gly3-Gly2-Gly'-Orn3-Orn2-Orn'] forms unusually stable and rigid complexes with metal ions. The rigid structure of the metal-peptide complex results in excellent NMR spectroscopic resolution of the six amide hydrogens and the temperature and pH dependence of the amide hydrogen exchange rates have been measured (10-12). The structure of ferrichrome is illustrated in Fig. 1. The peptide backbone forms a type II 3 turn and the modified ornithyl side chains provide octahedral coordination of the iron. The amide protons of Gly-1 and -2 are completely exposed to solvent and exchange METHODSFor the ferrichrome study all atoms were treated explicitly, except CH, CH2, and CH3 groups, which were treated as extended atoms (14); the model contained 55 atoms. The empirical potential function employed has been described previously (17). The starting point for the analysis is a local minimum energy conformation. The x-ray structure of Van der Helm et al. (18) was energy minimized by 50 steepest descent steps followed by 50 Newton-Raphson steps. At convergence the minimum energy conformation dev...

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“…The local unfolding model (21,22) is one of the mechanisms by which the buried amide hydrogens can exchange. Two types of fluctuations can be envisioned.…”

Section: Correlated Motions In Ssimentioning

confidence: 99%

Carbon-13 NMR Method for the Detection of Correlated Hydrogen Exchange at Adjacent Backbone Peptide Amides and Its Application to Hydrogen Exchange in Five Antiparallel β Strands within the Hydrophobic Core of Streptomyces Subtilisin Inhibitor (SSI)

2005

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A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the 13 C NMR signals from peptide carbonyls. The line shape of the carbonyl 13 C i signal is influenced by differential H/D occupancy at the two adjacent amides: the H N i+1 (β-site) and the H N i (γ-site). At a carbon frequency of 75.4 MHz, the H→D isotope shifts on the 13 C signal are about 5-7 Hz for exchange at the β-site and 2 Hz or less for exchange at the γ-site. Since the effects at the two sites are additive, the time-dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites, but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k c ) and degree of correlated exchange (X βγ ) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective 13 C′ labeling at all valyl residues and one with selective 13 C′ labeling at all leucyl residues. This permitted the direct observation by one-dimensional 13 C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i+1 positions. The residues investigated were located in an α-helix and in a five-stranded antiparallel β-sheet. Samples of the two labeled proteins were prepared at various pH*values, and 13 C NMR spectra were collected at 50 °C prior to and at various times subsequent to transferring the sample from H 2 O to 2 H 2 O. Most of the slowly exchanging amides studied were intramolecular hydrogen bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding, but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k β and k γ ) were proportional to [OH − ], and the * To whom correspondence should be addressed. Telephone: +81-426-77-2544+81-426-77- . Fax: +81-426-77-2544 NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 27. Published in final edited form as:Biochemistry. . This enabled us to extract the pH-independent exchange rates (k β°, k γ°, and k c°) . In all cases in which correlated exchange could be measured, the observed sigmoidal pH-dependence of X βγ could be replicated roughly from the derived pH-independent rates. KeywordsStreptomyces subtilisin inhibitor; SSI; carbonyl 13 C NMR; stable isotope assisted NMR; H/D exchange; amide hydrogen; isotope shift; β-shift; γ-shift; correlated hydrogen exchangeThe internal fluctuations of protein molecules are deeply related to their structure and stability and contribute to their functions. Protein fluctuations have been ...

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Individual breathing reactions measured in hemoglobin by hydrogen exchange methods

Cited by 92 publications

References 23 publications

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

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

Normal mode paths for hydrogen exchange in the peptide ferrichrome

Carbon-13 NMR Method for the Detection of Correlated Hydrogen Exchange at Adjacent Backbone Peptide Amides and Its Application to Hydrogen Exchange in Five Antiparallel β Strands within the Hydrophobic Core of Streptomyces Subtilisin Inhibitor (SSI)

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