Membrane Assembly of Simple Helix Homo-Oligomers Studied via Molecular Dynamics Simulations

This is the publisher's version. Copyright 2007 by Elsevier.The assembly of simple transmembrane helix homo-oligomers is studied by combining a generalized Born implicit membrane model with replica exchange molecular dynamics simulations to sample the conformational space of various oligomerization states and the native oligomeric conformation. Our approach is applied to predict the structures of transmembrane helices of three proteins—glycophorin A, the M2 proton channel, and phospholamban—using only peptide sequence and the native oligomerization state information. In every case, the methodology reproduces native conformations that are in good agreement with available experimental structural data. Thus, our method should be useful in the prediction of native structures of transmembrane domains of other peptides. When we ignore the experimental constraint on the native oligomerization state and attempt de novo prediction of the structure and oligomerization state based only on sequence and simple energetic considerations, we identify the pentamer as the most stable oligomer for phospholamban. However, for the glycophorin A and the M2 proton channels, we tend to predict higher oligomers as more stable. Our studies demonstrate that reliable predictions of the structure of transmembrane helical oligomers can be achieved when the observed oligomerization state is imposed as a constraint, but that further efforts are needed for the de novo prediction of both structure and oligomeric state

Enabling microbial syringol conversion through structure-guided protein engineering

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds

Vibrational Frequency Shifts and Relaxation Rates for a Selected Vibrational Mode in Cytochrome c

The vibrational energy relaxation of a selected vibrational mode in cytochrome c—a C-D stretch in the terminal methyl group of Met80—has been studied using equilibrium molecular dynamics simulation and normal mode analysis methods. As demonstrated in the pioneering work of Romesberg and co-workers, isotopic labeling of the C-H (to C-D) stretch in alkyl side chains shifts the stretching frequency to the transparent region of the protein's density of states, making it an effective and versatile probe of protein structure and dynamics. Molecular dynamics trajectories of solvated cytochrome c were run at 300 K, and vibrational population relaxation times were estimated using the classical Landau-Teller-Zwanzig model and a number of semiclassical theories of resonant and two-phonon vibrational relaxation processes. The C-D stretch vibrational population relaxation time is estimated to be T(1) = 14–40 ps; the relatively close agreement between various semiclassical estimates of T(1) lends support to the applicability of those expressions. Normal mode calculations were used to identify the dominant coupling between the protein and C-D oscillator. All bath modes strongly coupled to the C-D stretch are in close proximity. Angle bending modes in the terminal methyl group of Met80 appear to be the most likely acceptor modes defining the mechanism of population relaxation of the C-D vibration