Distribution Of Conformational States Research Articles

NMR chemical shift pattern changed by ammonium sulfate precipitation in cyanobacterial phytochrome Cph1.

Phytochromes are dimeric biliprotein photoreceptors exhibiting characteristic red/far-red photocycles. Full-length cyanobacterial phytochrome Cph1 from Synechocystis 6803 is soluble initially but tends to aggregate in a concentration-dependent manner, hampering attempts to solve the structure using NMR and crystallization methods. Otherwise, the Cph1 sensory module (Cph1Δ2), photochemically indistinguishable from the native protein and used extensively in structural and other studies, can be purified to homogeneity in >10 mg amounts at mM concentrations quite easily. Bulk precipitation of full-length Cph1 by ammonium sulfate (AmS) was expected to allow us to produce samples for solid-state magic-angle spinning (MAS) NMR from dilute solutions before significant aggregation began. It was not clear, however, what effects the process of partial dehydration might have on the molecular structure. Here we test this by running solid-state MAS NMR experiments on AmS-precipitated Cph1Δ2 in its red-absorbing Pr state carrying uniformly 13C/15N-labeled phycocyanobilin (PCB) chromophore. 2D 13C–13C correlation experiments allowed a complete assignment of 13C responses of the chromophore. Upon precipitation, 13C chemical shifts for most of PCB carbons move upfield, in which we found major changes for C4 and C6 atoms associated with the A-ring positioning. Further, the broad spectral lines seen in the AmS 13C spectrum reflect primarily the extensive inhomogeneous broadening presumably due to an increase in the distribution of conformational states in the protein, in which less free water is available to partake in the hydration shells. Our data suggest that the effect of dehydration process indeed leads to changes of electronic structure of the bilin chromophore and a decrease in its mobility within the binding pocket, but not restricted to the protein surface. The extent of the changes induced differs from the freezing process of the solution samples routinely used in previous MAS NMR and crystallographic studies. AmS precipitation might nevertheless provide useful protein structure/functional information for full-length Cph1 in cases where neither X-ray crystallography nor conventional NMR methods are available.

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

Sampling Issues in Atomistic Simulations of Peptides at Cyclohexane/Water and POPC/Water Interfaces

A large number of proteins and peptides interact with membranes to perform various biological activities such as signal transduction, transportation and antimicrobial activity. Characterizing the interactions of individual amino acids with lipid bilayers is an important step towards understanding lipid-protein interactions. The Wimley-White hydrophobicity scale pentapeptides WLXLL, where X can be any of the 20 amino-acid, adsorb at the POPC/water interface. Due to their small size and availability of experimental data, these peptides are suitable for studying lipid-peptide interactions using computational methods. We report atomistic simulations of Wimley-White peptides in bulk water and at cyclohexane/water and POPC/water interfaces. Various properties, such as end-to-end distance autocorrelations, the distribution of conformational states (based on dihedral angles) and the probability distributions of the center of mass of the side chains are measured. Using equilibrium molecular dynamic (MD) simulations, we were able to obtain good estimates of these properties only in bulk water. Even ∼600 ns molecular dynamic simulations at the POPC/water interface did not yield converged results. We also compare the sampling obtained using MD simulations with that of enhanced sampling methods such as Replica exchange MD simulations.These simulations highlight the sampling issues associated with simulation studies of unstructured peptides, which are relevant to a wide range of lipid-protein simulations including studies of antimicrobial peptides and peptide aggregation near membranes.

Site-Specific Methionine Oxidation and Calmodulin Molecular Dynamics

We have examined the structural consequences of methionine (Met) oxidation in the calcium signaling protein calmodulin (CaM) using molecular dynamics simulations. Protein oxidation by reactive oxygen species (ROS) is a critical element of cell function, but in the context of oxidative stress, has been implicated in disease progression and biological aging. Calmodulin function is intimately tied to muscle redox state through methionine oxidation by ROS and reduction by methionine sulfoxide reductases. Our goal is to bridge our understanding of muscle dysfunction and protein oxidation with atomic-level insights into site-specific methionine oxidation and calmodulin structural dynamics. We have carried out multiple 100 ns molecular dynamics simulations of explicitly solvated calmodulin starting from the calcium bound (1cll) and apo (1cfc) crystal structures. Results from preliminary simulations suggest that calcium bound CaM is structurally insensitive to methionine oxidation, while methionine oxidation in apo CaM causes considerable changes in the distribution of conformational states. Our work is a component of a larger study in which spectroscopic distance measurements and nuclear magnetic resonance experiments are being carried out for site-specifically oxidized CaM in both the calcium bound and apo biochemical states. We expect that our in silico results will bring atomic-level insight to spectroscopic measurements, and will be integral to creating a more complete model for oxidation-induced changes in calmodulin structural dynamics. Further, we anticipate that our results will be applicable to the many biological and pharmaceutical contexts in which a detailed understanding of protein oxidation, function and structure relationships is sought. This work is supported by NIH (2R37AG026160-06) and the Minnesota Supercomputing Institute.

Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle.

Binding of Zn(2+) to the endogenous Zn(2+) binding site in the human dopamine transporter leads to potent inhibition of [(3)H]dopamine uptake. Here we show that mutation of an intracellular tyrosine to alanine (Y335A) converts this inhibitory Zn(2+) switch into an activating Zn(2+) switch, allowing Zn(2+)-dependent activation of the transporter. The tyrosine is part of a conserved YXX Phi trafficking motif (X is any residue and Phi is a residue with a bulky hydrophobic group), but Y335A did not show alterations in surface targeting or protein kinase C-mediated internalization. Despite wild-type levels of surface expression, Y335A displayed a dramatic decrease in [(3)H]dopamine uptake velocity (V(max)) to less than 1% of the wild type. In addition, Y335A showed up to 150-fold decreases in the apparent affinity for cocaine, mazindol, and related inhibitors whereas the apparent affinity for several substrates was increased. However, the presence of Zn(2+) in micromolar concentrations increased the V(max) up to 24-fold and partially restored the apparent affinities. The capability of Zn(2+) to restore transport is consistent with a reversible, constitutive shift in the distribution of conformational states in the transport cycle upon mutation of Tyr-335. We propose that this shift is caused by disruption of intramolecular interactions important for stabilizing the transporter in a conformation in which extracellular substrate can bind and initiate transport, and accordingly that Tyr-335 is critical for regulating isomerization between discrete states in the transport cycle.

The structural distribution of cooperative interactions in proteins: analysis of the native state ensemble.

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids "sense" the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins (lambda repressor fragment 6-85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.

Time-resolved electrochromism associated with the formation of quinone anions in the Rhodobacter sphaeroides R26 reaction center.

The bacterial photosynthetic reaction center contains bacteriochlorophyll (Bchl) and bacteriopheophytin (Bph) cofactors that provide natural probes of electrostatic fields within this protein. We have examined the electrochromic responses of these cofactors, resolved during the lifetimes of the quinone anion states, P+QA-QB and P+QAQB-, and measured as a function of temperature. These measurements provide information on the time-dependent variation in electrostatic field strength on the Bchl and Bph cofactors. Measurements in the near-infrared absorbance bands have revealed the following. First, the QA-QB-->QAQB- electron transfer rate is found to be heterogeneous, consisting of at least two distinct kinetic components. At room temperature, we find a previously unresolved fast kinetic component with a reaction time of 25-40 microseconds, depending upon the preparation, that accounts for approximately 25% of the total reaction yield. The major component was identified with a reaction time of 210-240 microseconds. Below -20 degrees C, QA-QB-->QAQB- electron transfer shows distributed kinetics. The temperature-dependent conversion from biphasic to distributed kinetics suggests that there is a thermal averaging of conformational substates around two reaction center configurations. Interestingly, direct excitation of the Bph with 532 nm light at low temperatures appears to alter the electron transfer kinetics, possibly by inducing a change in the distribution of conformational states. The reaction kinetics were found to be sensitive to the addition of ethylene glycol, which is likely to reflect an osmolarity effect. Second, time-dependent absorption changes of the Bchl and Bph cofactors are found to be kinetically decoupled. The rapid responses of the Bph bands are interpreted to reflect electron transfer, while the slower responses of the Bchl are interpreted to reflect slower relaxation events, possibly including proton uptake. Finally, we find that the electrochromic response and QA-QB-->QAQB- electron transfer to be sensitive to the preparative state of the reaction center, reflecting differences in quinone binding for reaction centers in different states of purification.

Distribution of conformational states as common source of g-and A-strain in the ESR spectra of proteins and glasses

Additional experimental evidence on the structural and dynamical similarity between proteins and glasses is provided by the analysis conducted on the ESR spectra, at different temperatures, of some copper proteins and of some Cu++-doped glasses formed by water and a second component (NaOH, DMSO, alcohols). All the spectra taken at temperatures below ∼ 210 K display a significant g- and A-strain that consists in a progressive broadening and decrese in the intensity of the copper hyperfine lines as the order of mI increases (I = 3/2 is the Cu nuclear spin). These spectral features are taken into account by using a theoretical model which attributes the random distribution of the electric ligand fields around the copper ions to a distribution of the conformational substate energies both in the proteins and in the amorphous materials.