Population Shuffling of Protein Conformations

Motions play a vital role in the functions of many proteins. Discrete conformational transitions to excited states, happening on timescales of hundreds of microseconds, have been extensively characterized. On the other hand, the dynamics of the ground state are widely unexplored. Newly developed high‐power relaxation dispersion experiments allow the detection of motions up to a one‐digit microsecond timescale. These experiments showed that side chains in the hydrophobic core as well as at protein–protein interaction surfaces of both ubiquitin and the third immunoglobulin binding domain of protein G move on the microsecond timescale. Both proteins exhibit plasticity to this microsecond motion through redistribution of the populations of their side‐chain rotamers, which interconvert on the picosecond to nanosecond timescale, making it likely that this “population shuffling” process is a general mechanism.     

Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis

Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of side chain (13)C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cgamma resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (nonproton) gamma-substituents and with gauche conformational orientations of gamma-substituents. The (13)C shift/conformation correlations are most apparent for Cgamma carbons but also can be observed at positions further from the backbone. Intraresidue steric conflict leads to a correlation between upfield-shifted side chain (13)C resonances and statistically lower probabilities in surveys of protein side chain conformation. Illustrative applications to the DNA pol lambda lyase domain and to dihydrofolate reductase are discussed. In the latter case, (13)C shift analysis indicates that the conformation of the remote residue V119 on the betaF-betaG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. It is anticipated that (13)C shift data for protein sidechains can provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, deltaCdelta1-deltaCdelta2, on both chi1 and chi2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.     

Computational design of thermostabilizing D-amino acid substitutions

Judicious incorporation of D-amino acids in engineered proteins confers many advantages such as preventing degradation by endogenous proteases and promoting novel structures and functions not accessible to homochiral polypeptides. Glycine to D-alanine substitutions at the carboxy termini can stabilize α-helices by reducing conformational entropy. Beyond alanine, we propose additional side chain effects on the degree of stabilization conferred by D-amino acid substitutions. A detailed, molecular understanding of backbone and side chain interactions is important for developing rational, broadly applicable strategies in using D-amino acids to increase protein thermostability. Insight from structural bioinformatics combined with computational protein design can successfully guide the selection of stabilizing D-amino acid mutations. Substituting a key glycine in the Trp-cage miniprotein with D-Gln dramatically stabilizes the fold without altering the protein backbone. Stabilities of individual substitutions can be understood in terms of the balance of intramolecular forces both at the α-helix C-terminus and throughout the protein.     

Importance of chirality and reduced flexibility of protein side chains: a study with square and tetrahedral lattice models

Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our results suggest that side chain entropy may be an important factor contributing to the formation of alpha helices for compact conformations.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Functional assessment of “in vivo” and “in silico” mutations in the guanine binding site of RNase T1: A DFT study

Experimentally, the functional assessment of amino acid side chains in proteins is carried out by comparing parameters such as binding constants for the wild‐type protein and a mutant protein in which the considered side chain is deleted. In the present study, we apply a density functional theory (DFT) methodology to obtain changes in binding energy upon mutations in the enzyme ribonuclease T₁. Mutant structures were either taken directly from crystallographic data (“in vivo”) allowing for conformational changes upon mutation, or derived from the wild‐type (“in silico”). Excluding entropic contributions, the computed interaction energy changes upon mutation in vivo correlate qualitatively well with experimental binding free energy changes. In contrast, the in silico approach does not perform as well, especially for residues that contribute largely to binding. Subsequently, we assessed the applicability of the in vivo approach by analyzing the functional cooperativity between pairs of side chains. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004     

Re-evaluation of the model-free analysis of fast internal motion in proteins using NMR relaxation

NMR spin relaxation retains a central role in the characterization of the fast internal motion of proteins and their complexes. Knowledge of the distribution and amplitude of the motion of amino acid side chains is critical for the interpretation of the dynamical proxy for the residual conformational entropy of proteins, which can potentially significantly contribute to the entropy of protein function. A popular treatment of NMR relaxation phenomena in macromolecules dissolved in liquids is the so-called model-free approach of Lipari and Szabo. The robustness of the mode-free approach has recently been strongly criticized and the remarkable range and structural context of the internal motion of proteins, characterized by such NMR relaxation techniques, attributed to artifacts arising from the model-free treatment, particularly with respect to the symmetry of the underlying motion. We develop an objective quantification of both spatial and temporal asymmetry of motion and re-examine the foundation of the model-free treatment. Concerns regarding the robustness of the model-free approach to asymmetric motion appear to be generally unwarranted. The generalized order parameter is robustly recovered. The sensitivity of the model-free treatment to asymmetric motion is restricted to the effective correlation time, which is by definition a normalized quantity and not a true time constant and therefore of much less interest in this context. With renewed confidence in the model-free approach, we then examine the microscopic distribution of side chain motion in the complex between calcium-saturated calmodulin and the calmodulin-binding domain of the endothelial nitric oxide synthase. Deuterium relaxation is used to characterize the motion of methyl groups in the complex. A remarkable range of Lipari-Szabo model-free generalized order parameters are seen with little correlation with basic structural parameters such as the depth of burial. These results are contrasted with the homologous complex with the neuronal nitric oxide synthase calmodulin-binding domain, which has distinctly different thermodynamic origins for high affinity binding.     

Collective motions of myosin head derived from backbone molecular dynamics and combination with X‐ray solution scattering data

Collective modes of myosin head, S1, were derived from molecular dynamics trajectories of a simplified protein model, backbone model. Because the model requires only the positions of C_α‐atoms, large proteins are tractable, even when the amino acid sequence and the side‐chain orientations are unknown. The S1 is a large molecule and only the C_α‐atomic positions have been experimentally determined. In the simulation, large flipping motions of the extended α‐helical C‐terminal tail of S1 were found only in the largest and second largest amplitude collective modes, which were approximately perpendicular to each other. A few collective modes other than the first and second largest ones largely deformed the actin binding site of S1. The pair‐distance distribution, obtained from the X‐ray solution scattering, suggests a large conformational change of S1, isolated in solution without binding to actin filament, during the ATP hydrolysis. The modeling of the large conformational change was done with using the collective modes, and showed that the second mode mainly contributed to the large conformational change. The modeling procedure, introduced here, can be easily generalized for various experimental data, and applicable to the collective modes obtained from an all‐atom simulation. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1983–1994, 2001     

Predicting three-dimensional structures of transmembrane domains of β-barrel membrane proteins

β-Barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They are important for pore formation, membrane anchoring, and enzyme activity. These proteins are also often responsible for bacterial virulence. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank. We have developed a computational method for predicting structures of the transmembrane (TM) domains of β-barrel membrane proteins. Based on physical principles, our method can predict structures of the TM domain of β-barrel membrane proteins of novel topology, including those from eukaryotic mitochondria. Our method is based on a model of physical interactions, a discrete conformational state space, an empirical potential function, as well as a model to account for interstrand loop entropy. We are able to construct three-dimensional atomic structure of the TM domains from sequences for a set of 23 nonhomologous proteins (resolution 1.8-3.0 ?). The median rmsd of TM domains containing 75-222 residues between predicted and measured structures is 3.9 ? for main chain atoms. In addition, stability determinants and protein-protein interaction sites can be predicted. Such predictions on eukaryotic mitochondria outer membrane protein Tom40 and VDAC are confirmed by independent mutagenesis and chemical cross-linking studies. These results suggest that our model captures key components of the organization principles of β-barrel membrane protein assembly.     

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in 1H‐Detected Solid‐State NMR Spectroscopy

¹H‐detection can greatly improve spectral sensitivity in biological solid‐state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of ¹H‐detection by the loss of aliphatic side‐chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for ¹H‐detected ssNMR, and it gives high quality spectra for both side‐chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent ¹H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.     

Side‐chain conformational changes during the thermoshrinking process: γ‐Ray polymerization and spectroscopic study of uncrosslinked poly(methacryloyl‐Ala‐OMe)

Poly(methacryloyl‐L ‐alanine‐methyl ester) (1) has an optically active side chain and consists of thermoshrinking hydrogels upon crosslinking. We synthesized an uncrosslinked polymer of 1 by the γ‐ray polymerization method. For the prepared polymer, variable‐temperature circular dichroism (CD) and ¹H NMR spectra were studied, and we found conformational changes in the optically active side chains during the thermally induced phase transition. Intense CD spectra reveal ordered conformation in the side chain of 1 below the phase transition temperature (∼28 °C). A well‐resolved ¹H NMR spectrum of 1 at 0 °C shows that the conformational angles in the polymer side chain are fixed at low‐energy minima. With increasing temperature, the frozen side chain starts rotating vigorously and takes an unordered orientation. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2671–2677, 2000     

Side-chain dynamics analysis of KE07 series

The significant improvement of KE07 series in catalytic activities shows the great success of computational design approaches combined with directed evolution in protein design. Understanding the protein dynamics in the evolutionary optimization process of computationally designed enzyme will provide profound implication to study enzyme function and guide protein design. Here, side chain squared generalized order parameters and entropy of each protein are calculated using 50 ns molecular dynamics simulation data in both apo and bound states. Our results show a correlation between the increase of side chain motion amplitude and catalytic efficiency. By analyzing the relationship between these two values, we find side chain squared generalized order parameter is linearly related to side chain entropy, which indicates the computationally designed KE07 series have similar dynamics property with natural enzymes.     

The effect of point mutation on the equilibrium structural fluctuations of ferric Myoglobin

The ultrafast equilibrium fluctuations of the Fe(III)-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.     

Thermodynamics of polymeric fluids – effect of volume on the configurational entropy of chain molecules

Various conformation‐dependent properties of chain molecules have been successfully treated within the rotational isomeric state approximation. The conformation entropy is one of such properties which can be readily defined by the partition function, the sum of all possible configurations of the chain. Flexible polymers often exhibit crystallization and in some cases liquid‐crystallization as well. In these first‐order transitions, changes in the spatial arrangement of polymer chains are considered to be a major factor involved. In order to explicitly determine the conformational contribution to the melting entropy, the latent entropy observed under the isobaric condition must be corrected for the volume change. The entropy separation involves a hypothetical assumption that the volume of the isotropic fluid may be compressed to that of the solid state without affecting the configurational part of the entropy of molecules. Finally thermodynamic significance of the conformation entropy in these transitions is emphasized on the basis of the critical studies of the entropy‐volume relation of chain molecules in the liquid state.     

Surprising Effects on the Conformational Entropy due to Nonrandom Distributions of Local Conformations Along Unperturbed Chains

Summary: Nonrandomness in the distribution of rotational isomeric states along a flexible unperturbed chain reduces its conformational entropy. Pairwise interdependence of the bonds is a necessary, but not sufficient, condition for a significant reduction. The reduction in conformational entropy from this source can be as severe as a factor of three. It is generally more severe for isotactic chains than for the syndiotactic chains constructed from the same monomer. Surprising effects are sometimes seen, such as the nearly identical reductions in conformational entropy for polydimethylsiloxane, a very flexible chain, and for poly(methyl methacrylate), a much stiffer chain.

Fractional difference in conformational entropy due to nonrandomness versus probability of helix in helix‐coil transition.     

Identifying Protein Conformational Dynamics Using Spin‐label ESR

Spin‐label electron spin resonance (ESR) has emerged as a powerful tool to characterize protein dynamics. One recent advance is the development of ESR for resolving dynamical components that occur or coexist during a biological process. It has been applied to study the complex structural and dynamical aspects of membranes and proteins, such as conformational changes in protein during translocation from cytosol to membrane, conformational exchange between equilibria in response to protein‐protein and protein‐ligand interactions in either soluble or membrane environments, protein oligomerization, and temperature‐ or hydration‐dependent protein dynamics. As these topics are challenging but urgent for understanding the function of a protein on the molecular level, the newly developed ESR methods to capture individual dynamical components, even in low‐populated states, have become a great complement to other existing biophysical tools.     

Randomizing the Unfolded State of Peptides (and Proteins) by Nearest Neighbor Interactions between Unlike Residues

To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we combined vibrational and 2D NMR spectroscopy to obtain the conformational distributions of selected “GxyG” host–guest peptides in aqueous solution: GDyG, GSyG, GxLG, GxVG, where x/y=A, K, L, V. Large changes of conformational propensities were observed due to nearest‐neighbor interactions, at variance with the isolated pair hypothesis. We found that protonated aspartic acid and serine lose their above‐the‐average preference for turn‐like structures in favor of polyproline II (pPII) populations in the presence of neighbors with bulky side chains. Such residues also decrease the above‐the‐average pPII preference of alanine. These observations suggest that the underlying mechanism involves a disruption of the hydration shell. Thermodynamic analysis of ³J(H^N,H^α) (T) data for each x,y residue reveals that modest changes in the conformational ensemble masks larger changes of enthalpy and entropy governing the pPII?β equilibrium indicating a significant residue dependent temperature dependence of the peptides’ conformational ensembles. These results suggest that nearest‐neighbor interactions between unlike residues act as conformational randomizers close to the enthalpy–entropy compensation temperature, eliminating intrinsic biases in favor of largely balanced pPII/β dominated ensembles at physiological temperatures.     

Conformational preferences of model aliphatic diamides: Effect of the methyl side group on the polymethylene segment

The conformational preferences of N‐[3‐(acetylamino)‐2‐methyltrimethylene] acetamide and N‐[5‐(acetylamino)‐(S)‐2‐methylpentamethylene]acetamide were investigated using ab initio computational methods. These are model compounds of a number of substituted nylons‐m,n containing a methyl side group in the diamine unit. A comparison with the results obtained for their linear analogues have allowed us to determine the influence of the methyl side group on the conformational preferences of aliphatic diamides. Furthermore, the conformations compatible with the electron and X‐ray diffraction data recently obtained for the related substituted nylons‐m,n were identified.