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

Sw, Englander; Jj, Englander

doi:10.1016/0076-6879(94)32041-1

Cited by 38 publications

(24 citation statements)

References 37 publications

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“…After some exchange-out (chase) time, label on allosterically insensitive sites (same HX rate in both forms) is largely lost, but it is retained on allosterically sensitive sites, which are slowed in deoxy Hb. Thus, the exchange-in͞exchange-out sequence produces a sample with label selectively placed on functionally sensitive sites (6,31). HX measurement then reveals the number of sensitive residues and their HX rates in both forms.…”

Section: Methodsmentioning

confidence: 99%

“…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)(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.

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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%

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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.

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202

191

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“…For Ͼ40 years, peptide amide hydrogen-exchange techniques have been used to study the thermodynamics of protein conformational change and the mechanisms of protein folding (12,13). More recently, they have proven to be increasingly powerful methods by which protein dynamics, domain structure, regional stability, and function can be studied (14,15).…”

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confidence: 99%

“…The chemical mechanisms of the exchange reactions are understood, and several well defined factors can profoundly alter exchange rates (12,(39)(40)(41). One of these factors is the extent to which a particular exchangeable hydrogen is exposed (accessible) to water.…”

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confidence: 99%

“…In effect, each amide's exchange rate in a native protein directly and precisely reports solvent accessibility to it, thereby revealing the protein's thermodynamic stability on the scale of individual amino acids. Measurement of the exchange rates of a protein's amides can therefore allow direct identification and localization of structured͞unstructured regions of the protein; unstructured regions are those where substantial contiguous stretches of primary sequence exhibit the maximal possible exchange rates, which is indicative of complete and continuous solvation of the amide hydrogens in such segments (12,13). With its highthroughput capabilities, DXMS can rapidly localize disorder within crystallographic targets by using a minimum of protein sample.…”

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Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

Pantazatos

Kim

Klock

et al. 2004

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

142

109

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Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen͞high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins. When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

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