Dynamics of the quaternary conformational change in trout hemoglobin

Hofrichter, James; Henry, Eric R.; Szabó, Attila; Murray, Lionel P.; Ansari, Anjum; Jones, Colleen M.; Coletta, Massimo; Falcioni, Giancarlo; Brunori, Maurizio; Eaton, William A.

doi:10.1021/bi00240a031

Cited by 40 publications

(45 citation statements)

References 41 publications

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“…The measurements were made with a nanosecond spectrometer as described (10,13,14). The instrument is based on two Q-switched Nd/YAG (yttrium/aluminum garnet) lasers producing 10-ns (full width at halfmaximum) pulses.…”

Section: Methodsmentioning

confidence: 99%

Fast events in protein folding initiated by nanosecond laser photolysis.

Jones

Henry

et al. 1993

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

331

403

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Initiation of protein folding by light can dramaticafly improve the time resolution of kinetic studies. Here we present an example ofan optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are doser to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.Protein folding generally occurs in two phases, one rapid and one slow. The rapid phase is the collapse of the unfolded polypeptide into a compact structure and the formation of secondary structure. The slow phase is the subsequent, often multistep rearrangement to the native conformation (1-7). Because of the limited time resolution (milliseconds) of conventional stopped-flow mixing experiments, the complete time course of protein folding has not yet been observed. A dramatic improvement in time resolution would result if protein folding could be initiated by light.Here we present an example of an optically triggered folding reaction. We take advantage of the fact that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide (CO) to the covalently attached heme group in the unfolded state. The folding reaction can thus be triggered by photodissociating the CO complex. In this study, we used time-resolved absorption spectroscopy with nanosecond lasers to monitor binding events at the heme (8-11). Although rebinding of CO prevents the complete formation of the native conformation, the rapid recovery of the sample permits repetitive photolysis and therefore the acquisition of high signal/noise transient spectra for investigating submillisecond events. Simulations of the multiwavelength data with kinetic models were carried out to generate the spectra of intermediates, as well as the rate constants connecting them. We find that before folding begins there is transient binding of both nonnative and native ligands from the unfolded polypeptide chain on a microsecond time scale. MATERIALS AND METHODSTye VI horse heart cytochrome c from Sigma was purified by ion-exchange chromatography using carboxymethyl-cellulose (Whatman) (12), reduced with sodium dithionite anaerobically, and separated anaerobically from the sodium dithionite by gel filtrat...

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Section: Methodsmentioning

confidence: 99%

Fast events in protein folding initiated by nanosecond laser photolysis.

Jones

Henry

et al. 1993

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

331

403

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“…In such a hybrid molecule O 2 is not distributed preferentially on one type of subunit, as observed in the T-state (40), but the structure is more reminiscent of one of the possible diligated intermediates within a scheme which does not obey the symmetry principle of the classical two-state model (41). Such an intermediate shows the structural characteristics of the species possibly involved in the transition path (35,(42)(43)(44), corresponding to a tetramer in a quaternary R-like conformation associated to a tertiary T-like structure (at least for some subunit).…”

Section: Energetics Of Interactions Between Allosteric Effectors and mentioning

confidence: 98%

Coupling of the Oxygen-linked Interaction Energy for Inositol Hexakisphosphate and Bezafibrate Binding to Human HbA0

Coletta¹,

Angeletti²,

Ascenzi³

et al. 1999

Journal of Biological Chemistry

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The energetics of signal propagation between different functional domains (i.e. the binding sites for O 2 , inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA 0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to ؊3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O 2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO 2 ‫؍‬ 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T-and R-like tertiary conformation(s) within a quaternary R-like structure).Binding properties of gaseous ligands to human HbA 0 1 are markedly influenced by the so-called third components (in addition to HbA 0 and heme's ligands), such as organic phosphates (e.g. 2,3-bisphosphoglycerate and inositol hexakisphosphate (IHP)), protons, and chloride ions (1-4), which interact with HbA 0 at sites topologically different (heterotropic sites) from the heme.These heterotropic functional effects have been previously investigated in thermodynamic (5, 6) and kinetic (7) terms, leading to the evidence of a network of interplays among different sites, such that linkage relationships can be put in evidence between different heterotropic ligands. However, a quantitative investigation of these effects has never been performed since the information is usually obtained through a differential influence of the non-heme binding properties for the deoxygenated and oxygenated derivatives of HbA 0 .We have recently reported on the IHP binding properties to some heme ligated forms of ferrous HbA 0 in the presence of 0.1 M chloride, showing the existence of two binding sites for IHP with a similar affinity independently of the heme ligand (8). In addition, a recent thermodynamic study has been carried out on the interaction of IHP with oxygenated HbA 0 (9), leading to the evidence that the inter-relationship between protons and IHP is regulated by the pK a shift of three classes of residues in HbA 0 , and it is characterized by a proton-linked balancing between the enthalpic and entropic contribution. Furthermore, in the last few years particular interest has been addressed toward the effect of bezafibrate (BZF) (see Fig. 1) on the O 2 -binding properties of HbA 0 , and mostly on the original observation that its effect is potentiated by the presence of organic phosphates (10, 11), indicating that the two heterotropic ligands bind to different sites (12). This observation is interesting, since it underlies the existence...

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“…1 show that the activation enthalpies and entropies for the To -* Ro transition are much more similar to the equilibrium enthalpies and entropies than the corresponding values for the Row-To transition. That is, the thermodynamic properties of the transition state are much more similar to those of the R quaternary conformation than to those of the T quaternary conformation (4).t…”

mentioning

confidence: 95%

“…In a recent study on trout hemoglobin, time-resolved absorption spectroscopy was used to determine the activation parameters for the R -* T transition of the molecule with no ligands bound (4). At 20'C, the conformational change was found to occur with a rate constant of 5 x 104 sec1, and with an activation energy, Ea, of 8.0 kcal/mol (1 cal = 4.184 J).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin.

Eaton

Henry

Hofrichter

1991

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

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The transition state for the R = T quaternary conformational change of hemoglobin has thermodynamic properties much closer to those of the R conformation than to those of the T conformation. This finding is based on a comparison of activation and equilibrium enthalpy and entropy changes and on the observation of a linear free energy relationship between quaternary rate and equilibrium constants. A previous theoretical study [Janin, J. & Wodak, S. J. (1985) Biopolymers 24, 509-526], using a highly simplified energy function, suggests that the R-like transition state is the result of a reaction pathway with the maximum buried surface area

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Dynamics of the quaternary conformational change in trout hemoglobin

Cited by 40 publications

References 41 publications

Fast events in protein folding initiated by nanosecond laser photolysis.

Fast events in protein folding initiated by nanosecond laser photolysis.

Coupling of the Oxygen-linked Interaction Energy for Inositol Hexakisphosphate and Bezafibrate Binding to Human HbA0

Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin.

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