Protein folding in a reverse micelle environment: the role of confinement and dehydration.

Characterization of the molecular interactions that stabilize the folded state of proteins including hydrogen bond formation, solvation, molecular crowding, and interaction with membrane environments is a fundamental goal of theoretical biophysics. Inspired by recent experimental studies by Gai and co-workers, we have used molecular dynamics simulations to explore the structure and dynamics of the alanine-rich AKA(2) peptide in bulk solution and in a reverse micelle environment. The simulated structure of the reverse micelle shows substantial deviations from a spherical geometry. The AKA(2) peptide is observed to (1) remain in a helical conformation within a spherically constrained reverse micelle and (2) partially unfold when simulated in an unconstrained reverse micelle environment, in agreement with experiment. While aqueous solvation is found to stabilize the N- and C-termini random coil portions of the peptide, the helical core region is stabilized by significant interaction between the nonpolar surface of the helix and the aliphatic chains of the AOT surfactant. The results suggest an important role for nonpolar peptide-surfactant and peptide-lipid interactions in stabilizing helical geometries of peptides in reverse micelle environments.

Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations.

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.

Structure of the cytoplasmic domain of erythrocyte band 3 hereditary spherocytosis variant P327R: band 3 Tuscaloosa.

Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electron-electron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in beta-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.

Dipolar coupling between nitroxide spin labels: the development and application of a tether-in-a-cone model.

A tether-in-a-cone model is developed for the simulation of electron paramagnetic resonance spectra of dipolar coupled nitroxide spin labels attached to tethers statically disordered within cones of variable halfwidth. In this model, the nitroxides adopt a range of interprobe distances and orientations. The aim is to develop tools for determining both the distance distribution and the relative orientation of the labels from experimental spectra. Simulations demonstrate the sensitivity of electron paramagnetic resonance spectra to the orientation of the cones as a function of cone halfwidth and other parameters. For small cone halfwidths (< approximately 40 degrees ), simulated spectra are strongly dependent on the relative orientation of the cones. For larger cone halfwidths, spectra become independent of cone orientation. Tether-in-a-cone model simulations are analyzed using a convolution approach based on Fourier transforms. Spectra obtained by the Fourier convolution method more closely fit the tether-in-a-cone simulations as the halfwidth of the cone increases. The Fourier convolution method gives a reasonable estimate of the correct average distance, though the distance distribution obtained can be significantly distorted. Finally, the tether-in-a-cone model is successfully used to analyze experimental spectra from T4 lysozyme. These results demonstrate the utility of the model and highlight directions for further development.

Solution structure of the cytoplasmic domain of erythrocyte membrane band 3 determined by site-directed spin labeling.

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.