Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange

Englander, Joan J.; Louie, Godfrey; McKinnie, Russell E.; Englander, S. Walter

doi:10.1006/jmbi.1998.2278

Cited by 22 publications

(28 citation statements)

References 64 publications

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“…When all of the allosterically sensitive interactions are lost in liganded R-state Hb, each set of sensitive hydrogens moves as a group to a new rate, faster by 9-fold (32), 30-fold (37), and 750-fold (24). When specific mutations or chemical modifications are used to interdict individual allosterically sensitive interactions, the hydrogens in each affected HX set move to a common faster rate (19,24,38,39). The same result occurs when particular heme sites are selectively liganded (40).…”

Section: Discussionmentioning

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

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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: Discussionmentioning

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.

Self Cite

202

191

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“…Structural changes in complexes of vancomycin with peptide derivatives in water have provided clear-cut experimental evidence for tightening in strongly bound complexes (29), and similar signatures of structural tightening (and loosening) have been found in protein-ligand interactions (6,7,(13)(14)(15)(16)(17). Whitesides and coworkers (30) presented a study of the thermodynamics of binding of a vancomycin trimer with its corresponding trimeric ligand and showed that the enthalpy of binding was additive.…”

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confidence: 89%

Cooperativity in the self-assembly of porphyrin ladders

Camara-Campos

Hunter

Tomàs

2006

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

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Cooperativity is a general feature of intermolecular interactions in biomolecular systems, but there are many different facets of the phenomenon that are not well understood. Positive cooperativity stabilizes a system as progressively more interactions are added, and the origin of the beneficial free energy may be entropic or enthalpic in origin. An ''enthalpic chelate effect'' has been proposed to operate through structural tightening that improves all of the functional group interactions in a complex, when it is more strongly bound. Here, we present direct calorimetric evidence that no such enthalpic effects exist in the cooperative assembly of supramolecular ladder complexes composed of metalloporphyrin oligomers coordinated to bipyridine ligands. The enthalpic contributions of the individual coordination interactions are 35 kJ⅐mol ؊1 and constant over a range of free energies of self-assembly of ؊35 to ؊111 kJ⅐mol ؊1 . In rigid well defined systems of this type, the enthalpies of individual interactions are additive, and no enthalpic cooperative effects are apparent. The implication is that in more flexible, less well defined systems such as biomolecular assemblies, the enthalpy contributions available from specific functional group interactions are well defined and constant parameters.cooperative phenomena ͉ enthalpy-entropy compensation ͉ supramolecular chemistry ͉ weak interactions C ooperativity is of fundamental importance for understanding molecular recognition processes in chemistry and biology. If two molecules bearing one complementary binding site interact to give a complex of stability ⌬G, then two molecules bearing n complementary binding sites generally interact to give a complex with stability larger than n⌬G. This phenomenon is known as positive cooperativity. The factors that are responsible for cooperative intermolecular interactions can be divided in two groups: entropic factors relating to the loss of motion of the molecules and enthalpic factors due to the reinforcement of the bonds. The simplest formulation of the entropic factors is the chelate effect: an interaction working in isolation must pay the entropic cost of bringing two molecules together, but when multiple interactions are made, this price is paid only once, so additional interactions make a bigger contribution to stability than the first one (1-3). The entropic term also includes any change in the internal rotations and vibrations of the molecules. Enthalpic contributions to cooperative binding can come from secondary functional group interactions, conformational changes, or polarization of the interacting groups. However, enthalpy and entropy are intimately related, so an increase in the enthalpic driving force for complexation has a direct impact on the mobility of the molecules involved in the complex. For weak intermolecular interactions, the entropy lost on formation of the first interaction is only a fraction of the total possible loss, because the molecules retain a good deal of independent mobility. As the number of ...

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“…The native-state ensemble of a protein consists of an equilibrium of conformational states and its redistribution upon ligand binding has been suggested to impinge on cooperativity 11,12,[33][34][35] . Fluctuations of the structures allow the amide sites to sample a manifold of conformations, some of which permit exchange with the solvent.…”

Section: Effect Of Camp Binding On the Native-state Ensemblementioning

confidence: 99%

Dynamically driven protein allostery

Popovych

Sun

Ebright

et al. 2006

Nat Struct Mol Biol

585

628

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Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, a pronounced conformational entropic penalty is incurred by the second cAMP binding that is entirely responsible for the observed cooperativity. The results provide strong support of the existence of purely dynamics-driven allostery.Allosteric regulation is widely used in biological systems as a very effective mechanism to control protein activity [1][2][3][4] . Although a variety of allosteric systems have been studied for many decades, the mechanisms that underlie the communication of distant sites and energetically couple them remain largely elusive. According to the classical "mechanical" view allosteric interactions are mediated by a series of discrete conformational changes that alter protein structure [5][6][7] . Allosteric regulation within or across protein subunits requires that spatially separated sites be energetically coupled. Because allostery is fundamentally thermodynamic in nature, long-range communication may be mediated not only from a change in the mean conformation (enthalpic contribution) but also from a change in the dynamic fluctuations about the mean position (entropic contribution) 8,9 . The current view of allostery tends to explain the phenomenon only in pure structural terms, neglecting the important role of protein motions. To the other extreme, allosteric processes could, in theory, proceed solely through alteration in protein dynamics in the absence of conformational changes 10 . While there is increasing evidence suggesting a role of conformational dynamics in cooperative ligand binding processes 1,[11][12][13][14][15][16][17] , it has not been known whether dynamic phenomena can dominate allosteric mechanisms in proteins. Here, we present direct experimental evidence that allostery can be driven solely by changes in protein dynamics.Typically, studies of systems exhibiting homotropic cooperativity have focused on comparison of unliganded beginning and ligand-bound end states. However, the key conformational state with the potential to provide detailed insight into the mechanisms that underpin the allosteric process is the intermediate state. These states cannot be isolated in positively cooperative proteins because the singly liganded intermediates are poorly populated. In contrast, negatively *To whom correspondence should be addressed: babis@an...

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Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange

Cited by 22 publications

References 64 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

Cooperativity in the self-assembly of porphyrin ladders

Dynamically driven protein allostery

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