High Frequency Dynamics in Hemoglobin Measured by Magnetic Relaxation Dispersion

Abstract

The magnetic relaxation dispersion profiles for formate, acetate, and water protons are reported for aqueous solutions of hemoglobin singly and doubly labeled with a nitroxide and mercury(II) ion at cysteines at beta-93. Using two spin labels, one nuclear and one electron spin, a long intramolecular vector is defined between the two beta-93 positions in the protein. The paramagnetic contributions to the observed 1H spin-lattice relaxation rate constant are isolated from the magnetic relaxation dispersion profiles obtained on a dual-magnet apparatus that provides spectral density functions characterizing fluctuations sensed by intermoment dipolar interactions in the time range from the tens of microseconds to approximately 1 ps. Both formate and acetate ions are found to bind specifically within 5 angstroms of the beta-93 spin-label position and the relaxation dispersion has inflection points corresponding to correlation times of 30 ps and 4 ns for both ions. The 4-ns motion is identified with exchange of the anions from the site, whereas the 30-ps correlation time is identified with relative motions of the spin label and the bound anion in the protein environment close to beta-93. The magnetic field dependence of the paramagnetic contributions in both cases is well described by a simple Lorentzian spectral density function; no peaks in the spectral density function are observed. Therefore, the high frequency motions of the protein monitored by the intramolecular vector defined by the electron and nuclear spin are well characterized by a stationary random function of time. Attempts to examine long vector fluctuations by employing electron spin and nuclear spin double-labeling techniques did not yield unambiguous characterization of the high frequency motions of the vector between beta-93 positions on different chains.

Water-proton-spin–lattice-relaxation dispersion of paramagnetic protein solutions

“…This long range 3-dimensional contribution adds a non-logarithmic term to the magnetic field dependence. We note that a nearly logarithmic dependence has been previously reported for spin-labeled hemoglobin which was analyzed in terms of a distribution of local sticking times [13]. While a distribution of interfacial water interactions is likely, the effects of 2-dimensinoal diffusive exploration may be at least as important.…”

“…As a consequence, the spectral density functions characterizing this motion are logarithmic in the Larmor frequency [11]. The logarithmic magnetic field dependence of the spin-lattice relaxation rate has been observed in high surface area inorganic systems, but recent data suggest that these observations are more general and found in protein systems as well [12; 13]. X For a protein in solution, the spectral density formulation is slightly different from that of a lamellar solid structure and we explore the solution case in the present work to determine whether the change in dimensionality of diffusive exploration at the interface may account for the apparent increase in the electron-nuclear dipolar coupling strength.…”

Dimensionality of Diffusive Exploration at the Protein Interface in Solution

“…Distributions of surface dynamics are likely and supported by simulations [11, 12, 15–18]. Several have suggested that surface molecule residence times are distributed by a power law, a suggestion consistent with 1 H MRD on paramagnetic hemoglobin [38]. However, the distribution required in such approaches is both broad and strongly asymmetric.…”

Molecular Dynamics Simulations of Hemoglobin A in Different States and Bound to DPG: Effector-Linked Perturbation of Tertiary Conformations and HbA Concerted Dynamics

“…Undoubtedly, the next challenge that must be addressed is the experimental monitoring of HbA dynamics. The experimental characterization of protein fluctuations is, however, often complicated by the derivatizations required to obtain the experimental signal, as shown, for example, by the recent magnetic relaxation dispersion profiles recently obtained for bovine cyanoHb (97). In this context, time-resolved Laue experiments come to mind, such as those reported for Mb, which described the complex landscape of Mb structural dynamics after ligand dissociation (25,26).…”