Publications

Raman spectra were recorded experimentally and calculated theoretically for bithiophene, terthiophene, and quaterthiophene samples as a function of excitation polarization. Distinct spectral signatures were assigned and correlated to the molecular/unit cell orientation as determined by X-ray diffraction. The ability to predict molecular/unit cell orientation within organic crystals using polarized Raman spectroscopy was evaluated by predicting the unit cell orientation in a simulated terthiophene crystal given a random set of simulated polarized Raman spectra. Polarized Raman spectroscopy offers a promising tool to quickly and economically determine the unit cell orientation in known organic crystals and crystalline thin films. Implications of our methodologies for studying individual molecule conformations are discussed.

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations.

The Horner method was used to synthesize random copolymers of poly(2-methoxy-5-(2′-ethylhexyloxy)-p-phenylene vinylene) (MEH-PPV) that incorporated different backbone-directing monomers. Single-molecule polarization absorption studies of these new polymers demonstrate that defects that preserve the linear backbone of PPV-type polymers assume the highly anisotropic configurations found in defect-free MEH-PPV. Rigid defects that are bent lower the anisotropy of the single chain, and saturated defects that provide rotational freedom for the chain backbone allow for a wide variety of possible configurations. Molecular dynamics simulations of model defect PPV oligomers in solution demonstrate that defect-free and linearly defected oligomers remain extended while the bent and saturated defects tend toward more folded, compact structures.

Despite the high density within a typical protein fold, the ensemble of sterically permissible side-chain repackings is vast. Here, we examine the extent of this variability that survives energetic biases due to van der Waals interactions, hydrogen bonding, salt bridges, and solvation. Monte Carlo simulations of an atomistic model exhibit thermal fluctuations among a diverse set of side-chain arrangements, even with the peptide backbone fixed in its crystallographic conformation. We have quantified the torsional entropy of this native-state ensemble, relative to that of a noninteracting reference system, for 12 small proteins. The reduction in entropy per rotatable bond due to each kind of interaction is remarkably consistent across this set of molecules. To assess the biophysical importance of these fluctuations, we have estimated side-chain entropy contributions to the binding affinity of several peptide ligands with calmodulin. Calculations for our fixed-backbone model correlate very well with experimentally determined binding entropies over a range spanning more than 80 kJ/(mol·308 K).

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Aβ42, α-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the “sensitive regions” for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.

Protein aggregation is associated with a variety of pathological conditions, including Alzheimer s and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.

Paramagnetic metal centers [such as Fe^III found within ferriprotoporphyrin IX heme (FPIX)] exert through space effects on the relaxation rate of nearby proton spins that depend critically on the metal−proton distance. We have measured these effects for all protons of several antimalarial drugs that bind to FPIX by systematically varying the drug:heme molar ratio in high field NMR experiments. These measurements allow us to determine precise FPIX Fe−drug H distances for the solution structures of noncovalent complexes formed between FPIX μ-oxo dimers and the antimalarial drugs chloroquine (CQ), quinine (QN), and quinidine (QD). Using these distances, we then performed distance restraint calculations to determine the lowest-energy solution structures of these complexes. Structures were solved for neutral, monoprotic (+1), and diprotic (+2) forms of the drugs. Analysis of these structures allows us to visualize for the first time the stereospecific differences between QN and QD binding to FPIX and the differences in populations of QN and QD solution structures upon changes in digestive vacuolar pH for drug resistant malarial parasites [Dzekunov, S. M., et al. (2000) Mol. Biochem. Parasitol. 110, 107−124]. The data indicate a previously unrecognized key role for the CQ aliphatic chain in stabilizing FPIX−CQ complexes, and suggest how lengthening or shortening the chain might perturb stability. We also define FPIX:drug stoichiometries of 2:1 for the complexes formed at physiological FPIX concentrations, in contrast to the 4:1 and 5:1 stoichiometries previously determined at higher FPIX concentrations [Dorn, A., et al. (1998) Biochem. Pharmacol. 55, 727−736]. These atomic resolution antimalarial drug−heme structures should help elucidate how these drugs inhibit formation of hemozoin during metabolism of heme within the malarial parasite Plasmodium falciparum and assist ongoing development of strategies for circumventing antimalarial drug resistance.

Recently, we measured a more acid digestive vacuolar pH for drug resistant Plasmodium falciparum [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press; Ursos LMB, Dzekunov S, Roepe PD. Mol Biochem Parasitol 2000;in press]. We suggested this acidification contributes to drug resistance via the profound effects that pH has on the solubility of unpolymerized heme found in the vacuole (ferriprotoporphyrin IX μ oxo dimers). In this report, we measure how FPIX concentration, time, NaCl concentration, and several antimalarial drugs affect FPIX pH dependent solubility. Aggregation is essentially instantaneous below pH 5.3, but at vacuolar pH previously measured for HB3 parasites [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press] can increase to several minutes as NaCl is lowered. As FPIX is decreased, the midpoint of the pH dependent solubility curve shifts to higher values. Addition of antimalarial drugs also increases the midpoint of the pH dependent FPIX solubility curve, with the net shift proportional to the relative affinity of the drug for FPIX. Surprisingly, however, for all drugs tested shifts of essentially identical magnitude are found at all drug: FPIX molar ratios inspected, spanning eight orders of magnitude (to as low as 0.0000001:1). This suggests that changes in pH dependent FPIX solubility by addition of antimalarial drugs is via previously unrecognized drug/FPIX nucleation phenomena. These data could have important implications for understanding the role of previously observed changes in pHvac [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press; Ursos LMB, Dzekunov S, Roepe PD. Mol Biochem Parasitol 2000;in press] upon development of antimalarial drug resistance.