Allosteric Intermediates in Hemoglobin. 1. Nanosecond Time-Resolved Circular Dichroism Spectroscopy

Björling, Sofie C.; Goldbeck, Robert A.; Paquette, S. J.; Milder, Steven J.; Kliger, David S.

doi:10.1021/bi952247s

Cited by 45 publications

(45 citation statements)

References 45 publications

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“…In the case of aqueous COMb, the relaxation is largely complete within tens of picoseconds but with some residual components persisting well beyond 10 ns (23,28,48,50,51,62,64,82,88,(101)(102)(103). In dramatic contrast, for COHbA, the tertiary relaxation does not begin until several nanoseconds subsequent to photodissociation (48,57,58,(103)(104)(105)(106). Consequently, slowing down the relaxation will, in the case of Mb, eventually allow the unrelaxed photoproduct to persist to beyond nanoseconds, whereas for HbA, the photoproduct already persists beyond nanoseconds.…”

Section: Discussionmentioning

confidence: 99%

Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin

Samuni

Dantsker

Khan

et al. 2002

Journal of Biological Chemistry

108

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We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. The method allows for direct comparison of functional and spectroscopic properties of equilibrium and non-equilibrium populations under the same temperature and viscosity conditions. The results implicate tertiary structure changes that include the proximal heme environment in the mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of (Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the degree of separation between the F helix and the heme is assigned as the conformational coordinate that modulates both this frequency and the innermost barrier controlling CO rebinding. A comparison with the behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are qualitatively similar to the tertiary changes that occur within both the R and T quaternary states. Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix. Myoglobin (Mb)1 continues to be used as a model protein system for investigations into how structure, dynamics, and reactivity interconnect to give rise to functionality. Recently there has been a renewed interest in myoglobin based on several new developments. From a functional point of view there are indications and suggestions that, apart from its physiological importance in facilitating oxygen diffusion, myoglobin has functions arising from its ability to participate in catalytic multisubstrate reactions involving such molecules as NO, O 2 , and H 2 O 2 (1-3). In addition the presence of apolar intra-protein substrate docking sites (the so-called Xe cavities) (4, 5) have recently been linked both to these newly proposed functions and to the kinetic patterns associated with geminate and bimolecular ligand binding (3, 6 -13).These new developments highlight fundamental biophysical issues that have not been fully addressed despite the extensive research effort that has been directed toward myoglobin. The issue that we address in this work relates to ligand reactivity as a function of conformation. Several studies show that myoglobin can adopt different tertiary conformations. In particular there is evidence that the equilibrium distribution of tertiary conformations is different for the deoxy (ferrous five coordinate) and CO derivatives of Mb. X-ray crystallographic studies show that there is a small clam shell-like rotation of the E and F helices in going from deoxy to COMb (14) as well as adjustments in the positioning of residues in the distal heme pocket (8 -10, 15, 16) that may or may not be related to the clam shell motion. Recent time-resolved x-ray crystallographic stud...

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Section: Discussionmentioning

confidence: 99%

Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin

Samuni

Dantsker

Khan

et al. 2002

Journal of Biological Chemistry

108

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“…11,12 More recently, timeresolved UV resonance Raman (TR-UVRR), timeresolved circular dichroism, and time-resolved magnetic circular dichroism have been used to complement optical absorption and resonance Raman data. [13][14][15] TR-UVRR studies, besides identifying fast submicrosecond relaxations connected to the E and F helices clamshell rotation and to the breaking/reformation of interhelical hydrogen bonds, 16 have indicated a stepwise quaternary transition in Hb in which a fast relaxation in 2 μs is followed by a slower one in 20-50 μs. TR-UVRR studies attributed the fast relaxation to the formation of quaternary H-bonds at the "hinge region" of the α 1 β 2 interface and suggested that dimer rotation is almost completed already at these short times; the slower 20-μs relaxation was attributed to formation of contacts in the "switch region" of the α 1 β 2 interface and to tertiary relaxation at the hemes.…”

Section: Introductionmentioning

confidence: 99%

Unveiling the Timescale of the R–T Transition in Human Hemoglobin

Cammarata

Levantino

Wulff

et al. 2010

Journal of Molecular Biology

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Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with timeresolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place ∼ 2 μs after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-μs step observed with time-resolved optical spectroscopy corresponds to a small and localized structural change.

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“…These studies show that conformational changes that occur before 1 s are purely tertiary, whereas those occurring later include quaternary changes as well (6,(14)(15)(16)(17). Therefore, the ligation-linked tertiary changes can, in principle, be elucidated at the atomic level by determining the structure of a short-time photoproduct of HbCO by using x-ray crystallography.…”

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confidence: 99%

“…Insight into pure tertiary structural changes in Hb has been gained form time-resolved spectroscopic measurements with the use of the photodissociation of CO-liganded Hb (HbCO), to generate unliganded Hb (deoxyHb), which then evolves from the original R conformation toward the equilibrium T conformation before it fully recombines with the CO (14)(15)(16)(17). These studies show that conformational changes that occur before 1 s are purely tertiary, whereas those occurring later include quaternary changes as well (6,(14)(15)(16)(17).…”

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confidence: 99%

Direct observation of photolysis-induced tertiary structural changes in hemoglobin

Adachi

Park

Tame

et al. 2003

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

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Human Hb, an ␣2␤2 tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded Rstate human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the ␣ subunit than in the ␤ subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state ␤ heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.A llosteric transitions allow rapid regulation of protein function in biological systems. There is a wealth of structural data regarding the end states of allosteric transitions of various proteins (1), but very little is known about how they work. A typical example of allosteric regulation is the cooperative oxygen binding by human Hb. The Hb molecule is a heterotetramer consisting of two ␣ subunits and two ␤ subunits, ␣ 2 ␤ 2 , which are arranged as a dimer of ␣␤ dimers. Each subunit contains one heme group to which one oxygen molecule binds reversibly. The oxygen affinity of each subunit rises as the other hemes in the same tetramer become saturated with oxygen. The binding of oxygen by Hb is therefore cooperative, allowing efficient transport of oxygen in the blood. Crystal structures of fully unliganded tense (T)-state and fully liganded relaxed (R)-state Hb reveal multiple differences at both tertiary and quaternary levels (2-5), but, as is the case for most other allosteric proteins, through what mechanism these different structures impact ligand reactivity is still not well understood (for review, see ref. 6). An essential part of this question is understanding the mechanism of restraints on ligand binding in the T state, because the oxygen affinity of the R state is close to that of isolated ␣ and ␤ subunits. In the early 1970s, Perutz suggested that the low affinity of the T state is caused by tension in the iron-proximal His(F8) bond in the liganded T state (7), but several lines of evidence show that the ligand affinity of the ␣ and ␤ subunits is regulated by different mechanisms (8)(9)(10)(11)(12). Despite the wealth of data from x-ray crystallography, a means of defining the stereochemical basis unambiguously for the difference in oxygen affinity between the T and R states, which is only a few kcal͞mol, has not been determined (13). It is important to note that the larg...

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Allosteric Intermediates in Hemoglobin. 1. Nanosecond Time-Resolved Circular Dichroism Spectroscopy

Cited by 45 publications

References 45 publications

Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin

Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin

Unveiling the Timescale of the R–T Transition in Human Hemoglobin

Direct observation of photolysis-induced tertiary structural changes in hemoglobin

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