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

Abstract

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...

Ligand recombination and a hierarchy of solvent slaved dynamics: The origin of kinetic phases in hemeproteins

“…The present treatment represents a distillation and out growth of our recent work utilizing sol-gel encapsulation and glassy matrices to modulate protein dynamics in a way that provides a direct bridge between cryogenic and ambient domains Dantsker et al, 2005a;Dantsker et al, 2005b). We (Khan et al, 2000;Dantsker et al, 2002;Samuni et al, 2002;Dantsker et al, 2005a;Dantsker et al, 2005b) and others (Sottini et al, 2004;Sottini et al, 2005a;Sottini et al, 2005b;Sottini et al, 2005c) have shown that this approach of using high viscosity solvents and matrices exposes the dynamical events that give rise to the solution phase functional properties observed in solution at ambient temperatures. …”

“…These would include for Mb, the dynamics contributing to the relaxation of the initial photoproduct tertiary structure back to the equilibrium deoxy conformation and for hemoglobins the additional larger scale motions required for changes in quaternary structure. Both categories of dynamics can be greatly slowed within sol-gel matrices relative to the operative time scales of the C and D tiers of dynamics (Bettati and Mozarrelli, 1997;Das et al, 1999;Khan et al, 2000;Abbruzzetti et al, 2001;Dantsker et al, 2002;Samuni et al, 2002;Shibayama and Saigo, 2003;Samuni et al, 2004;Viappiani et al, 2004). Consequently, the above A, B, C and D scheme can be explored within the conformational boundaries of different trapped tertiary and quaternary structures that represent both equilibrium and nonequilibrium conformational distributions.…”

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

“…[5][6][7][8][9][10][11][12][13] In addition, tertiary and quaternary conformational changes in heme proteins, such as myoglobin (Mb) and hemoglobin (Hb), can be inhibited or dramatically slowed, which allows these proteins to be "trapped" and studied in non-equilibrium structural conformations. [14][15][16][17][18][19][20][21][22] The origin of the stability imparted by the silica sol-gel matrix is complex. Since the mean pore diameters in aged wet sol-gels (typically <10 nm) are comparable to the diameters of Mb and Hb, the pore walls could physically restrict protein motions.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…The possibility provided by silica gel encapsulation to reduce the rate of conformational changes by orders of magnitude, increasing the kinetic separation of tertiary and quaternary relaxations [57,58,[80][81][82][83][84][85][86][87][88][89][90][91][92], has been extensively exploited by several groups to make accessible to spectroscopic investigation species that are normally poorly populated, or metastable on the time scale of solution experiments. Not surprisingly, many of these studies concern hemoglobin (Hb), myoglobin and other heme binding proteins, since the availability of a sensitive, intrinsic chromophoric signal allowed to achieve an unprecedented level of detail in the comprehension of structure-dynamic-function relationships.…”

“…Since the quaternary structural change of encapsulated Hb is greatly slowed down, tertiary intermediate states along the R T transition could be observed by conventional spectroscopic methods such as UV resonance Raman spectroscopy [86,89,90]. In particular, it has been shown that after photolysis of HbCO encapsulated in the T state, both the E and F helices assume intermediate positions with respect to those that characterize the Hb equilibrium states.…”