Side-chain Transitions Research Articles

Near-Ultraviolet Circular Dichroism and Two-Dimensional Spectroscopy of Polypeptides.

A fully quantitative theory of the relationship between protein conformation and optical spectroscopy would facilitate deeper insights into biophysical and simulation studies of protein dynamics and folding. In contrast to intense bands in the far-ultraviolet, near-UV bands are much weaker and have been challenging to compute theoretically. We report some advances in the accuracy of calculations in the near-UV, which were realised through the consideration of the vibrational structure of the electronic transitions of aromatic side chains.

Dynamic elements and kinetics: Most favorable conformations of peptides in solution with measurements and simulations.

Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 310 helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 310 helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.

DichroCalc: Improvements in Computing Protein Circular Dichroism Spectroscopy in the Near-Ultraviolet

A fully quantitative theory connecting protein conformation and optical spectroscopy would facilitate deeper insights into biophysical and simulation studies of protein dynamics and folding. The web server DichroCalc (http://comp.chem.nottingham.ac.uk/dichrocalc) allows one to compute from first principles the electronic circular dichroism spectrum of a (modeled or experimental) protein structure or ensemble of structures. The regular, repeating, chiral nature of secondary structure elements leads to intense bands in the far-ultraviolet (UV). The near-UV bands are much weaker and have been challenging to compute theoretically. We report some advances in the accuracy of calculations in the near-UV, realized through the consideration of the vibrational structure of the electronic transitions of aromatic side chains. The improvements have been assessed over a set of diverse proteins. We illustrate them using bovine pancreatic trypsin inhibitor and present a new, detailed analysis of the interactions which are most important in determining the near-UV circular dichroism spectrum.

Recent advances in metathesis-derived polymers containing transition metals in the side chain.

This account critically surveys the field of side-chain transition metal-containing polymers as prepared by controlled living ring-opening metathesis polymerization (ROMP) of the respective metal-incorporating monomers. Ferrocene- and other metallocene-modified polymers, macromolecules including metal-carbonyl complexes, polymers tethering early or late transition metal complexes, etc. are herein discussed. Recent advances in the design and syntheses reported mainly during the last three years are highlighted, with special emphasis on new trends for superior applications of these hybrid materials.

Gating by tryptophan 73 exposes a cryptic pocket at the protein-binding interface of the oncogenic eIF4E protein.

Targeting protein-protein interacting sites for potential therapeutic applications is a challenge in the development of inhibitors, and this becomes more difficult when these interfaces are relatively planar, as in the eukaryotic translation initiation factor 4E (eIF4E) protein. eIF4E is an oncogene that is overexpressed in numerous forms of cancer, making it a prime target as a therapeutic molecule. We report here the presence of a cryptic pocket at the protein-binding interface of eIF4E, which opens transiently during molecular dynamics simulations of the protein in solvent water and is observed to be stable when solvent water is mixed with benzene molecules. This pocket can also be seen in the ensemble of structures available from the solution-state conformations of eIF4E. The accessibility of the pocket is gated by the side-chain transitions of an evolutionarily conserved tryptophan residue. It is found to be feasible for accommodating clusters of benzene molecules, which signify the plasticity and ligandability of the pocket. We also observe that the newly formed cavity provides a favorable binding environment for interaction of a well-recognized small molecule inhibitor of eIF4E. The occurrence of this transiently accessible cavity highlights the existence of a more pronounced binding groove in a region that has traditionally been considered to be planar. Together, the data suggest that an alternate binding cavity exists on eIF4E and could be exploited for the rational design and development of a new class of lead compounds against the protein.

Atomistic Characterization of the First Step of Calcium Pump Activation Associated with Proton Countertransport

The calcium pump [sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA)] transports Ca(2+) from the cytosol to the SR lumen at the expense of ATP hydrolysis and proton countertransport, thus playing a central role in Ca(2+) homeostasis and muscle contractility. Proton countertransport via deprotonation of transport site residue Glu309 is a critical first step in SERCA activation because it accelerates the E2-E1 structural transition. Previous studies have suggested that flipping of Glu309 toward the cytosol constitutes the primary mechanism for Glu309 deprotonation, but no conclusive data to support this hypothesis have been published. Therefore, we performed three independent 1 μs molecular dynamics simulations of the E2 state protonated at transport site residues Glu309, Glu771, and Glu908. Structural analysis and pKa calculations showed that Glu309 deprotonation occurs by an inward-to-outward side-chain transition. We also found that Glu309 deprotonation and proton countertransport occur through transient (~113 ps) water wires connecting Glu309 with the cytosol. Although both mechanisms are operational, we found that transient water wire formation, and not Glu309 flipping, is the primary mechanism for Glu309 deprotonation and translocation of protons to the cytosol. The outward-to-inward transition of protonated Glu309 and the presence of water wires suggest that protons from the cytosol might be passively transported to the lumen via Glu309. However, structural analysis indicates that passive SR proton leakage into the lumen unlikely occurs through Glu309 in the E2 state. These findings provide a time-resolved visualization of the first step in the molecular mechanism of SERCA activation and proton transport across the SR.

Enzyme architecture: optimization of transition state stabilization from a cation-phosphodianion pair.

The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in Km (2.8 kcal/mol effect) and a 41 000-fold decrease in kcat (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme–phosphodianion pairs.

Two-dimensional near-ultraviolet spectroscopy of aromatic residues in amyloid fibrils: a first principles study.

We report a first principles study of two dimensional electronic spectroscopy of aromatic side chain transitions in the 32-residue β-amyloid (Aβ(9-40)) fibrils in the near ultraviolet (250-300 nm). An efficient exciton Hamiltonian with electrostatic fluctuations (EHEF) algorithm is used to compute the electronic excitations in the presence of environmental fluctuations. The through-space inter- and intra-molecular interactions are calculated with high level quantum mechanics (QM) approaches, and interfaced with molecular mechanics (MM) simulations. Distinct two dimensional near ultraviolet (2DNUV) spectroscopic signatures are identified for different aromatic transitions, and the couplings between them. 2DNUV signals associated with the transition couplings are shown to be very sensitive to the change of residue-residue interactions induced by residue mutations. Our simulations suggest that 2DNUV spectra could provide a useful local probe for the structure and kinetics of fibrils.

Peptide Adsorption to Lipid Bilayers: Slow Processes Revealed by Linear Dichroism Spectroscopy

The adsorption and insertion kinetics for the association of two 34-residue cyclic peptides with phosphocholine membranes have been studied using circular and linear dichroism approaches. The two peptides studied are identical with the exception of two residues, which are both tyrosine in one of the peptides and tryptophan in the other. Both peptides adopt random coil conformations in solution in the absence of membranes and do not aggregate at concentrations below 20 μM. After addition to liposome dispersions, circular dichroism spectroscopy indicated that both peptides undergo an extremely rapid transformation to a β-conformation that remains unchanged throughout the remainder of the experiment. Linear dichroism (LD) spectroscopy was used to study the kinetics of membrane adsorption and insertion. The data were analyzed by nonlinear least squares approaches, leading to identification of a number of bound states and their corresponding LD spectra. Two pseudo-first order processes could be identified that were common to both peptides. The first occurred with a time constant of the order of 1 min and led to a bound state characterized by weak LD signals, with significant bands corresponding to the transitions of aromatic side chains. The second process occurred with an unusually long time constant of between 75 and 100 min, forming a state with considerably stronger positive LD absorbance in the far-ultraviolet region of the spectrum. For the tyrosine-substituted peptide, a third slow process with a long time constant (76 min) could also be delineated and was attributed to rearrangements of the peptide within the membrane.

Open Science Grid Study of the Coupling between Conformation and Water Content in the Interior of a Protein

Computational grids are a promising resource for modeling complex biochemical processes such as protein folding, penetration of gases or water into proteins, or protein structural rearrangements coupled to ligand binding. We have enabled the molecular dynamics program CHARMM to run on the Open Science Grid. The implementation is general, flexible, easily modifiable for use with other molecular dynamics programs and other grids and automated in terms of job submission, monitoring, and resubmission. The usefulness of grid computing was demonstrated through the study of hydration of the Glu-66 side chain in the interior of protein staphylococcal nuclease. Multiple simulations started with and without two internal water molecules shown crystallographically to be associated with the side chain of Glu-66 yielded two distinct populations of rotameric states of Glu-66 that differed by as much as 20%. This illustrates how internal water molecules can bias protein conformations. Furthermore, there appeared to be a temporal correlation between dehydration of the side chain and conformational transitions of Glu-66. This example demonstrated how difficult it is to get convergence even in the relatively simple case of a side chain oscillating between two conformations. With grid computing, we also benchmarked the self-guided Langevin dynamics method against the Langevin dynamics method traditionally used for temperature control in molecular dynamics simulations and showed that the two methods yield comparable results.

Charge-Transfer Transitions in the Vacuum-Ultraviolet of Protein Circular Dichroism Spectra

Circular dichroism (CD) is widely used in the structural characterization and secondary structure determination of proteins. The vacuum UV region (below 190 nm), where charge-transfer transitions have an influence on the CD spectra, can be accessed using synchrotron radiation circular dichroism (SRCD) spectroscopy. Recently, charge-transfer transitions in a conformationally diverse set of dipeptides have been characterized ab initio using complete active space self-consistent field calculations, and the relevant charge distributions have been parametrized for use in the matrix method for calculations of protein CD. Here, we present calculations of the vacuum UV CD spectra of 71 proteins, for which experimental SRCD spectra and X-ray crystal structures are available. The theoretical spectra are calculated considering charge-transfer and side chain transitions. This significantly improves the agreement with experiment, raising the Spearman correlation coefficient between the calculated and the experimental intensity at 175 nm from 0.12 to 0.79. The influence of the conformation on charge-transfer transitions is analyzed in detail, showing that the n --> pi* charge-transfer transitions are most important in alpha-helical proteins, whereas in beta strand proteins the pi --> pi* charge-transfer transition along the chain in the amino- to carboxy-end direction is most dominant.

Chromism in polythiophene derivatives

Thermochromism has been observed in some polythiophene derivatives both in the solid state and in solution. These optical effects are related to a planar/non-planar transition of the conjugated backbone. Various measurements have revealed a strong dependence of these chromic properties upon the nature, the position and the conformation of the substituents. These results seem to indicate that the twisting of the main chain could be driven by order-disorder “transitions” of the side chains. Order-disorder side-chain transition can be induced from different stimuli and this concept has led to the development of novel ionochromic polymers. Similar phenomena could also be induced through side chain cis-trans photoisomerisation (photochromism), or by side chains that would possess some affinity to chemical or biochemical molecules (affinity chromism).

Variation of free volume parameters of polyalkylmethacrylate derivatives in the range from 15 to 300 K probed by positron annihilation lifetime measurement

Positron annihilation lifetime measurements of PMMA, PEMA, PiPMA, PnPMA and PnBMA were performed in the temperature range between 15 and 300 K, where δ, γ and β relaxational transitions occur for these polyalkylmethacrylates. The variations of free volume size and content calculated from the longest lifetime component against temperature are correlated to the results obtained by dielectric, viscoelastic and dynamic mechanical relaxation data of the polymers. The variations of free volume sizes and contents, apparent free volume fraction and size distribution of the polyalkylmethacrylates are well correlated with the rotational transitions of side chains. The location of free volumes, in which positronium annihilates can be estimated near the vicinity of alkyl groups bound to oxygen atom of side ester chains.

Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long‐time molecular dynamics calculations in explicit water

The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using “realistic” potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.

Vacuum ultraviolet spectroscopy of the optical properties and electronic structure of seven poly(di-alkylsilanes)

We report the ultraviolet (UV) and vacuum ultraviolet (VUV) optical properties and electronic structure, up to 44 eV, of thin-film samples of seven poly(di-alkylsilanes) [alkyl = n-butyl, n-pentyl, i-hexyl, n-hexyl, n-octyl, and n-tetradecyl] with three types of SiSi backbone conformations: helical, planar zigzag, and trans-gauche- trans-gauche' (TGTG′). The backbone conformation determines the UV transitions, with helical materials exhibiting one near-UV absorption, while two UV transitions are seen for the two-phase materials containing both the helical and planar zigzag backbone conformations. The TGTG′ backbone exhibits a single UV absorption at 3.6 eV. At higher energies all materials show a prominent shoulder at 7.2 eV, with a doublet peak structure seen at ≈ 9 and ≈ 12 eV. The 7.2 eV transition is unaffected by the backbone conformation or alkyl substitution, while comparison with the electronic transitions in polyethylene shows that the high-energy double-peak structure corresponds to transitions in the hydrocarbon sidechains. The ≈ 12 eV transitions appear to shift to higher energy with increasing sidechain length and in tetradecyl these sidechain transitions are noticeably narrowed, suggesting unusual sidechain crystallization in poly(di-n-tetradecylsilane). A hierarchy of electronic transitions can be developed whereby the UV transitions arise in the Si backbone, at intermediate energies backbone to sidechain transitions are observed, while the high-energy transitions are of the alkyl sidechains. This hierarchy in the electronic transitions demonstrates the ability of VUV spectroscopy of the electronic structure to serve as a microscopic probe of the bonding and structure of polysilanes, for example providing detailed insight into the properties of the sidechains in these polymers.

Protein dynamics An overview on flash-photolysis over broad temperature ranges

Ligand binding kinetics to heme-proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein-specific susceptibility of the heme-iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash-photolysis measurements below ca. 180 K; we quantify it in terms of ‘intramolecular viscosity’, postulating that narrow low-temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high-frequency transitions of the amino acid side chains and low-frequency collective motions as a possible regulatory mechanism of protein dynamics.

Effects of anesthetics and dichlorodifluoromethane on the activities of glyceraldehyde-phosphate dehydrogenase and pectin methylesterase

Solutions of glucose-3-phosphate dehydrogenase (GPD) and pectin methylesterase (PME) were exposed to various anesthetics and dichlorodifluoromethane (F-12) to determine the abilities of these chemicals to inhibit enzyme activity. An aqeuous solution of PME was exposed to saturation levels of the test chemicals for 30 min at 21°. All test chemicals were inhibitory (measured after release of the test chemical) with propane being most inhibitory followed in order by F-12, cyclopropane, Ethrane (F 2HCOF 2CCHClF) and halothane (CF 3CHClBr). GPD was exposed to various concentrations of F-12 and halothane for various times at 0° and 33°. Halothane at 33° and a saturation concentration reduced the initial reaction velocity of GPD to zero after a 10-min exposure period. F-12 was somewhat less inhibitory than halothane, but inhibition in all instances was irreversible. Halothane was found to affect the circular dichroism and optical rotary dispersion spectra of GPD, with the magnitude of the changes generally increasing with treatment time. The observed changes were believed to arise from side-chain transitions of GPD.

Structure and properties of two plastic peptide triblock, ABA, copolymers of poly(γ-benzyl- l-glutamate), A, and poly(butadiene/acrylonitrile), B

Two prototype triblock (ABA) copolymers of poly[(γ-benzyl- l-glutamate) x (butadiene/acrylonitrile) y (γ-benzyl- l-glutamate) x] have been synthesized and characterized. They were prepared by reacting a primary amine capped butadiene/acrylonitrile (ATBN) polymer with the N- carboxy anhydride of γ-benzyl- l-glutamate. The copolymers were ∼38 000 (copolymer 1) and 74 000 (copolymer II) molecular weight. X-ray diffraction and Fourier Transform infrared spectroscopy of films cast from dioxane (preferential for PBLG) and chloroform (non-preferential) show the benzyl glutamate segments to be predominantly α-helical and disordered α-helical, respectively. Electron microscopy of osmium tetroxide strained films cast from dioxane revealed lamellar domain formation indicative of phase separation. The midblock butadiene layers were ∼150 Å thick while the alternating benzyl glutamate layers were 300 and 500 Å thick for copolymers I and II, respectively. Films cast from chloroform exhibit a nearly homogeneous morphology, indicative of considerable phase mixing. Dynamic mechanical spectroscopy of the copolymers also revealed a dependence on morphology. The side chain transition of the benzyl glutamate appeared as a single peak when the copolymers were cast from dioxane and a double peak when the copolymers were cast from chloroform.

Higher aliphatic aldehyde polymers, 8. Poly(undecanal)

AbstractCrystalline poly(undecanal) (2) was prepared by anionic polymerization of undecanal (1) with lithium tert‐butanolate in methylcyclohexane. As the aliphatic side chain length of the monomeric aldehyde increases, the polymerization becomes increasingly difficult and 2 could only be obtained if the temperature profile during the polymerization was carefully controlled. 2 of reasonable molecular weight was obtained in nearly 50% yield and was identified by IR and NMR spectroscopy. X‐ray diffraction measurements showed the polymer to be highly crystalline. The paraffinic side chains of 2 were found to be primarily responsible for the polymer crystallization and the polymer side chain transitions were found between 40 and 60°C. Normally, more than one endothermic peak was found by DSC in this region. 2 could be extended through an orifice to a coherent filament whose x‐ray diffraction pattern showed negligible orientation. In contrast, other aldehyde polymers with shorter side chains gave good fiber patterns on extrusion.

Solid state side chain transitions of poly(alpha-amino acids). I. Investigation by differential scanning calorimetry.

The differential scanning calorimetric behavior of a series of alpha-amino acid homopolymers and copolymers was investigated in the range of temperature from -80 to +130 degrees C. The following polymers have been examined: poly(N'-carbobenzoxy-L-lysine), poly(O-carbobenzoxy-L-tyrosine), random copolymers of O-carbobenzoxy-L-tyrosine and N'-carbobenzoxy-L-lysine, poly(Ngamma-carbobenzoxy-L-diaminobutyric acid), and poly(N sigma-carbobenzoxy-L-ornithine). Each sample exhibited a prominent glass-like transition between +30 and +60 degrees C with a specific heat increment deltacp of the order of 0.03-0.11 cal/(g degrees C). Endothermal peaks, developed by annealing, have been also been revealed. It is concluded that in poly(alpha-amino acids) side chain motions are capable of undergoing glass-like transition.