Kateri H. DuBay

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

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.

Impact of molecular symmetry on single-molecule conductance.

We have measured the single-molecule conductance of a family of bithiophene derivatives terminated with methyl sulfide gold-binding linkers using a scanning tunneling microscope based break-junction technique. We find a broad distribution in the single-molecule conductance of bithiophene compared with that of a methyl sulfide terminated biphenyl. Using a combination of experiments and calculations, we show that this increased breadth in the conductance distribution is explained by the difference in 5-fold symmetry of thiophene rings as compared to the 6-fold symmetry of benzene rings. The reduced symmetry of thiophene rings results in a restriction on the torsion angle space available to these molecules when bound between two metal electrodes in a junction, causing each molecular junction to sample a different set of conformers in the conductance measurements. In contrast, the rotations of biphenyl are essentially unimpeded by junction binding, allowing each molecular junction to sample similar conformers. This work demonstrates that the conductance of bithiophene displays a strong dependence on the conformational fluctuations accessible within a given junction configuration, and that the symmetry of such small molecules can significantly influence their conductance behaviors.

Calculation of Proteins’ Total Side-Chain Torsional Entropy and Its Influence on Protein–Ligand Interactions

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 x 308 K).

Polarized Raman spectroscopy of oligothiophene crystals to determine unit cell orientation.

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.

A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.