Pressure effects on the proximal heme pocket in myoglobin probed by Raman and near-infrared absorption spectroscopy

Galkin, Oleg; Buchter, Scott; Tabirian, Anna; Schulte, Alfons

doi:10.1016/s0006-3495(97)78304-8

Cited by 28 publications

(29 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the EPR spectrum of residue 87R1 in the F helix shows the appearance of an immobile component at 2 kbar, reflecting the onset of localized conformational exchange. This result is consistent with previous studies that identified rearrangements in the heme group upon pressurization that are transmitted to the F helix through the coordinating His93 (57,58). In addition, the pressure sensitivity of 106R1 in helix G reveals a small and highly localized compressibility that may occur in an otherwise rigid structure.…”

Section: Discussionsupporting

confidence: 92%

See 1 more Smart Citation

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Lerch

Horwitz²,

McCoy³

et al. 2013

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

View full text Add to dashboard Cite

Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF 2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.P roteins in solution are dynamic molecules, exhibiting conformational flexibility across a range of time and length scales (1). In addition to a well-ordered native state, conformational excursions to low-lying "excited states" may be required in protein function (2). For example, on a funnel-shaped energy landscape (3, 4), excited states have increased configurational entropy that may give rise to the promiscuous protein-protein interactions that define a protein interactome (5, 6). Structural changes involved in formation of the excited state may take a variety of forms, from rigid body motions of helices (7) to local unfolding of secondary structural elements (8). Despite their functional relevance, excited states are sparsely populated and may escape detection by standard spectroscopic techniques. Hydrostatic pressure apparently offers a solution to this problem by reversibly populating excited states of proteins, allowing for spectroscopic characterization (9-14). Pressure application is particularly useful because it shifts the relative population of preexisting conformational states while minimally affecting the conformational landscape itself (15).Assuming that pressure can populate excited states for study, the increased configurational entropy of such states presents a challenge to spectroscopic methods that aim to describe structure; depending on the intrinsic time scale of the method, either a population-weighted average structure or a heterogeneous ensemble is observed. The intrinsic time scale of con...

show abstract

Section: Discussionsupporting

confidence: 92%

“…This pressuredependent shift in population for 87R1 and 106R1 presumably is a reflection of local compressibility of the protein. For 87R1, this interpretation is consistent with crystallographic and near-IR/ Raman spectroscopic studies of holoMb at high pressure, which revealed flexibility in helix F (57,58).…”

Section: Resultssupporting

confidence: 86%

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Lerch

Horwitz²,

McCoy³

et al. 2013

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

View full text Add to dashboard Cite

show abstract

“…To the extent that the kinetic barrier is determined in part by a transition state that has the iron at least partially moved toward the heme plane, as is believed to be the case for the CO ligand (47, 113, 114), the binding rate will be responsive to proximal strain. This assessment is consistent with pressure studies on Mb in which increased pressure both increases the frequency of (Fe-His) and increases the geminate yield (115). In the case of the Mb samples, the difference in the kinetics arising from proximal effects would stem from the positioning of the F helix.…”

Section: Discussionsupporting

confidence: 85%

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

Samuni

Dantsker

Khan

et al. 2002

Journal of Biological Chemistry

108

View full text Add to dashboard Cite

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...

show abstract

“…The effects of pressure on hemeproteins have been the subject of numerous investigations. Optical absorption [16–21], fluorescence [22], FTIR [23–25], Raman [26], and NMR [27–29] spectroscopies, and laser flash photolysis [30–32] have all shown that pressures near 300 MPa leads to subtle local rearrangements of the protein structure and that some intermediate states preceding unfolding probably appear. Therefore, it is important to determine whether the modifications observed at the level of the active site of myoglobin [17,18,20,21,26–29] and the reorganization of the secondary structure with an alteration of the electrostatic and hydrogen‐bond array [23,24] are related to a change in the tertiary structure of the protein.…”

mentioning

confidence: 99%

High‐pressure effects on horse heart metmyoglobin studied by small‐angle neutron scattering

Loupiac

Bonetti

et al. 2002

European Journal of Biochemistry

View full text Add to dashboard Cite

Small-angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN 3 ) at pressures up to 300 MPa. Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performed using various concentrations of MbN 3 , show that the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa. The value of the second virial coefficient of the protein solution indicates that the interactions between the molecules are always strongly repulsive even if their magnitude decreases with increasing pressure. Taking advantage of the pressureinduced contrast variation, these experiments allow the partial specific volume of MbN 3 to be determined as a function of pressure. Its value decreases by 5.4% between atmospheric pressure and 300 MPa. In this pressure range the isothermal compressibility of hydrated MbN 3 is found to be almost constant. Its value is (1.6 ± 0.1) 10 )4 MPa )1.Keywords: myoglobin; pressure; SANS; partial volume; compressibility.The structure of proteins and their solvent interactions can be modified by temperature, pH or chemicals. The application of hydrostatic pressure to a protein solution also provides a manner to alter these physical properties [1][2][3][4]. Therefore, it is important to determine whether the modifications observed at the level of the active site of myoglobin [17,18,20,21,[26][27][28][29] and the reorganization of the secondary structure with an alteration of the electrostatic and hydrogen-bond array [23,24] are related to a change in the tertiary structure of the protein.In order to reply these questions we report here for the first time, the results of small-angle neutron scattering (SANS) experiments performed on myoglobin (Mb) under pressure. Quite generally, SANS can provide information about the partial volume of proteins, their interactions, their size and their conformation [33]. The scattering measurements were carried out in heavy water ( 2 H 2 O) at varying pressures up to 300 MPa as a function of protein concentration in order to determine the magnitude of the solute interactions and to allow for the elimination of their contribution to the forward scattered intensity and the apparent radius of gyration.Azidometmyoglobin (MbN 3 ) has a high stability. It was chosen for this study in order to avoid a mixture of aquo and hydroxy derivatives in metmyoglobin solutions or a contamination of either oxygen or carbon monoxide saturated myoglobin solutions by oxidized forms which could form under pressure [34].

show abstract

Pressure effects on the proximal heme pocket in myoglobin probed by Raman and near-infrared absorption spectroscopy

Cited by 28 publications

References 68 publications

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

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

High‐pressure effects on horse heart metmyoglobin studied by small‐angle neutron scattering

Contact Info

Product

Resources

About