Local Dynamics and Stability of Apocytochrome <i>b</i>₅₆₂ Examined by Hydrogen Exchange^†

Cytochrome b562 is a heme-binding protein consisting of four helices folded into a classic helix bundle motif. Though retaining much of the topology of the holoprotein, apocytochrome b562 displays physical features commonly associated with so-called protein molten globules. Here, the stability and dynamics of this “structured” molten globule are probed by examination of the dependence of its hydrogen exchange behavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b562 displays a limited dynamic range of hydrogen exchange rates and the analysis required the development of a quantitative approach. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle, with the N- and C-terminal helices being of independent and lower stability. The dependence of the global unfolding free energy upon denaturant concentration indicates the applicability of a binding model and explains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calorimetry and hydrogen exchange. These observations place a significant restraint upon the type of folding pathway that is operative for this protein and suggest that that the N- and C-terminal helices fold and unfold independently of the core of the molecule

A ¹³C Labeling Strategy Reveals a Range of Aromatic Side Chain Motion in Calmodulin

NMR relaxation experiments often require site-specific isotopic enrichment schemes in order to allow for quantitative interpretation. Here we describe a new labeling scheme for site-specific ¹³C–¹H enrichment of a single ortho position of aromatic amino acid side chains in an otherwise perdeuterated background by employing a combination of [4-¹³C]­erythrose and deuterated pyruvate during growth on deuterium oxide. This labeling scheme largely eliminates undesired contributions to ¹³C relaxation and greatly simplifies the fitting of relaxation data using the Lipari–Szabo model-free formalism. This approach is illustrated with calcium-saturated vertebrate calmodulin and oxidized flavodoxin from Cyanobacterium anabaena. Analysis of ¹³C relaxation in the aromatic groups of calcium-saturated calmodulin indicates a wide range of motion in the subnanosecond time regime

Mapping the Hydration Dynamics of Ubiquitin

The nature of water’s interaction with biomolecules such as proteins has been difficult to examine in detail at atomic resolution. Solution NMR spectroscopy is potentially a powerful method for characterizing both the structural and temporal aspects of protein hydration but has been plagued by artifacts. Encapsulation of the protein of interest within the aqueous core of a reverse micelle particle results in a general slowing of water dynamics, significant reduction in hydrogen exchange chemistry and elimination of contributions from bulk water thereby enabling the use of nuclear Overhauser effects to quantify interactions between the protein surface and hydration water. Here we extend this approach to allow use of dipolar interactions between hydration water and hydrogens bonded to protein carbon atoms. By manipulating the molecular reorientation time of the reverse micelle particle through use of low viscosity liquid propane, the T1ρ relaxation time constants of 1H bonded to 13C were sufficiently lengthened to allow high quality rotating frame nuclear Overhauser effects to be obtained. These data supplement previous results obtained from dipolar interactions between the protein and hydrogens bonded to nitrogen and in aggregate cover the majority of the molecular surface of the protein. A wide range of hydration dynamics is observed. Clustering of hydration dynamics on the molecular surface is also seen. Regions of long-lived hydration water correspond with regions of the protein that participate in molecular recognition of binding partners suggesting that the contribution of the solvent entropy to the entropy of binding has been maximized through evolution

Microscopic Insights into the NMR Relaxation-Based Protein Conformational Entropy Meter

Conformational entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of conformational entropy are necessary for an understanding of its role but have been difficult to obtain. An empirical method that utilizes changes in conformational dynamics as a proxy for changes in conformational entropy has recently been introduced. Here we probe the microscopic origins of the link between conformational dynamics and conformational entropy using molecular dynamics simulations. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain conformational entropy. These results tend to validate the use of experimentally accessible measures of methyl motionthe NMR-derived generalized order parametersas a proxy from which to derive changes in protein conformational entropy

Solution Structure of Horse Heart Ferricytochrome <i>c</i> and Detection of Redox-Related Structural Changes by High-Resolution ¹H NMR^†^,^‡

A model for the solution structure of horse heart ferricytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry−simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1671 distance constraints based on the observed magnitude of nuclear Overhauser effects and 58 torsion angle restraints based on the magnitude of determined J-coupling constants. The model incorporates six long-lived water molecules detected by pseudo-two-dimensional NOESY−TOCSY spectra. The all-residue root mean square deviation about the average structure is 0.33 ± 0.04 Å for the backbone N, Cα, and C‘ atoms and 0.83 ± 0.05 Å for all heavy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal structures of homologous c-type cytochromes though there are a number of significant differences in detailed aspects of the structure. Two of the three main helices display localized irregularities in helical hydrogen bonding resulting in bifurcation of main chain hydrogen bond acceptor carbonyls. The N- and C-terminal helices are tightly packed and display several interhelical interactions not seen in reported crystal models. To provide an independent measure of the accuracy of the model for the oxidized protein, the expected pseudocontact shifts induced by the spin 1/2 iron were compared to the observed redox-dependent chemical shift changes. These comparisons confirm the general accuracy of the model for the oxidized protein and its observed differences with the structure of the reduced protein. The structures of the reduced and oxidized states of the protein provide a template to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the cytochrome c molecule. For example, a redox-dependent reorganization of surface residues at the heme edge can be directly related to the redox behavior of the protein and thereby provides a previously undocumented linkage between structural change potentially associated with molecular recognition of redox partners and the fundamental parameters governing electron transfer

Validation of Protein Structure from Preparations of Encapsulated Proteins Dissolved in Low Viscosity Fluids

Validation of Protein Structure from Preparations of Encapsulated Proteins Dissolved in Low Viscosity Fluid

Protein Structure Determination by High-Resolution Solid-State NMR Spectroscopy: Application to Microcrystalline Ubiquitin

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D 13C−13C correlation spectroscopy under magic angle spinning conditions. High-resolution 13C spectra have been acquired from hydrated microcrystals of site-directed 13C-enriched ubiquitin. Interresidue carbon−carbon distance constraints defining the global protein structure have been evaluated from ‘dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 Å. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy

Comparison of ²H and ¹³C NMR Relaxation Techniques for the Study of Protein Methyl Group Dynamics in Solution

A comparison of 2H- and 13C-based NMR relaxation methods to characterize the dynamics of methyl groups in proteins is presented. Using human ubiquitin as a model system, the field dependence of carbon and deuterium relaxation parameters has been measured and used to probe the utility of various forms of the model-free formalism in revealing the underlying dynamics. We find that both approaches reveal the same overall dynamical features provided that suitable parametrization and model-free spectral densities are employed. It is found that the original and extended model-free formalisms yield different descriptions of the methyl group dynamics and that the extended version is more appropriate for the analysis of carbon relaxation. Because of the inherent differences in the types of information that 2H and 13C offer, deuterium methods appear to provide robust access to methyl symmetry axis order with the least amount of data, while carbon methods provide more robust access to model-free parameters defining the time scale of methyl rotation and methyl symmetry axis motion

Characterization of Cetyltrimethylammonium Bromide/Hexanol Reverse Micelles by Experimentally Benchmarked Molecular Dynamics Simulations

Encapsulation of small molecules, proteins, and other macromolecules within the protective water core of reverse micelles is emerging as a powerful strategy for a variety of applications. The cationic surfactant cetyl­trimethyl­ammonium bromide (CTAB) in combination with hexanol as a cosurfactant is particularly useful in the context of solution NMR spectroscopy of encapsulated proteins. Small-angle X-ray and neutron scattering is employed to investigate the internal structure of the CTAB/hexanol reverse micelle particle under conditions appropriate for high-resolution NMR spectroscopy. The scattering profiles are used to benchmark extensive molecular dynamics simulations of this reverse micelle system and indicate that the parameters used in these simulations recapitulate experimental results. Scattering profiles and simulations indicate formation of homogeneous solutions of small approximately spherical reverse micelle particles at a water loading of 20 composed of ∼150 CTAB and 240 hexanol molecules. The 3000 waters comprising the reverse micelle core show a gradient of translational diffusion that reaches that of bulk water at the center. Rotational diffusion is slowed relative to bulk throughout the water core, with the greatest slowing near the CTAB headgroups. The 5 Å thick interfacial region of the micelle consists of overlapping layers of Br^– enriched water, CTAB headgroups, and hexanol hydroxyl groups, containing about one-third of the total water. This study employs well-parametrized MD simulations, X-ray and neutron scattering, and electrostatic theory to illuminate fundamental properties of CTAB/hexanol reverse micelle size, shape, partitioning, and water behavior