Christopher D. Snow

Absolute comparison of simulated and experimental protein-folding dynamics

Protein folding is difficult to simulate with classical molecular dynamics. Secondary structure motifs such as α-helices and β-hairpins can form in 0.1–10 µs (ref. 1), whereas small proteins have been shown to fold completely in tens of microseconds. The longest folding simulation to date is a single 1-µs simulation of the villin headpiece; however, such single runs may miss many features of the folding process as it is a heterogeneous reaction involving an ensemble of transition states. Here, we have used a distributed computing implementation to produce tens of thousands of 5–20-ns trajectories (700 µs) to simulate mutants of the designed mini-protein BBA5. The fast relaxation dynamics these predict were compared with the results of laser temperature-jump experiments. Our computational predictions are in excellent agreement with the experimentally determined mean folding times and equilibrium constants. The rapid folding of BBA5 is due to the swift formation of secondary structure. The convergence of experimentally and computationally accessible timescales will allow the comparison of absolute quantities characterizing in vitro and in silico (computed) protein folding.

Simulation of Folding of a Small Alpha-helical Protein in Atomistic Detail using Worldwide- distributed Computing

By employing thousands of PCs and new worldwide-distributed computing techniques, we have simulated in atomistic detail the folding of a fast-folding 36-residue alpha-helical protein from the villin headpiece. The total simulated time exceeds 300 micros, orders of magnitude more than previous simulations of a molecule of this size. Starting from an extended state, we obtained an ensemble of folded structures, which is on average 1.7A and 1.9A away from the native state in C(alpha) distance-based root-mean-square deviation (dRMS) and C(beta) dRMS sense, respectively. The folding mechanism of villin is most consistent with the hydrophobic collapse view of folding: the molecule collapses non-specifically very quickly ( approximately 20ns), which greatly reduces the size of the conformational space that needs to be explored in search of the native state. The conformational search in the collapsed state appears to be rate-limited by the formation of the aromatic core: in a significant fraction of our simulations, the C-terminal phenylalanine residue packs improperly with the rest of the hydrophobic core. We suggest that the breaking of this interaction may be the rate-determining step in the course of folding. On the basis of our simulations we estimate the folding rate of villin to be approximately 5micros. By analyzing the average features of the folded ensemble obtained by simulation, we see that the mean folded structure is more similar to the native fold than any individual folded structure. This finding highlights the need for simulating ensembles of molecules and averaging the results in an experiment-like fashion if meaningful comparison between simulation and experiment is to be attempted. Moreover, our results demonstrate that (1) the computational methodology exists to simulate the multi-microsecond regime using distributed computing and (2) that potential sets used to describe interatomic interactions may be sufficiently accurate to reach the folded state, at least for small proteins. We conclude with a comparison between our results and current protein-folding theory.

A family of thermostable fungal cellulases created by structure-guided recombination

SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63 °C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63 °C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15 °C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.

Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli

2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

Using path sampling to build better Markovian state models: Predicting the folding rate and mechanism of a tryptophan zipper beta hairpin

We propose an efficient method for the prediction of protein folding rate constants and mechanisms. We use molecular dynamics simulation data to build Markovian state models (MSMs), discrete representations of the pathways sampled. Using these MSMs, we can quickly calculate the folding probability (P(fold)) and mean first passage time of all the sampled points. In addition, we provide techniques for evaluating these values under perturbed conditions without expensive recomputations. To demonstrate this method on a challenging system, we apply these techniques to a two-dimensional model energy landscape and the folding of a tryptophan zipper beta hairpin.

Native-like Mean Structure in the Unfolded Ensemble of Small Proteins

The nature of the unfolded state plays a great role in our understanding of proteins. However, accurately studying the unfolded state with computer simulation is difficult, due to its complexity and the great deal of sampling required. Using a supercluster of over 10,000 processors we have performed close to 800 micros of molecular dynamics simulation in atomistic detail of the folded and unfolded states of three polypeptides from a range of structural classes: the all-alpha villin headpiece molecule, the beta hairpin tryptophan zipper, and a designed alpha-beta zinc finger mimic. A comparison between the folded and the unfolded ensembles reveals that, even though virtually none of the individual members of the unfolded ensemble exhibits native-like features, the mean unfolded structure (averaged over the entire unfolded ensemble) has a native-like geometry. This suggests several novel implications for protein folding and structure prediction as well as new interpretations for experiments which find structure in ensemble-averaged measurements.

Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

We studied the microsecond folding dynamics of three β hairpins (Trp zippers 1–3, TZ1–TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLSaa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1–TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.

Direct calculation of the binding free energies of FKBP ligands

Direct calculations of the absolute free energies of binding for eight ligands to FKBP protein were performed using the Fujitsu BioServer massively parallel computer. Using the latest version of the general assisted model building with energy refinement (AMBER) force field for ligand model parameters and the Bennett acceptance ratio for computing free-energy differences, we obtained an excellent linear fit between the calculated and experimental binding free energies. The rms error from a linear fit is 0.4 kcal/mol for eight ligand complexes. In comparison with a previous study of the binding energies of these same eight ligand complexes, these results suggest that the use of improved model parameters can lead to more predictive binding estimates, and that these estimates can be obtained with significantly less computer time than previously thought. These findings make such direct methods more attractive for use in rational drug design.

SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability

A quantitative linear model accurately (R2 = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 °C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 °C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55–56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.

A diverse family of thermostable cytochrome P450s created by recombination of stabilizing fragments

Thermostable enzymes combine catalytic specificity with the toughness required to withstand industrial reaction conditions. Stabilized enzymes also provide robust starting points for evolutionary improvement of other protein properties. We recently created a library of at least 2,300 new active chimeras of the biotechnologically important cytochrome P450 enzymes. Here we show that a chimeras thermostability can be predicted from the additive contributions of its sequence fragments. Based on these predictions, we constructed a family of 44 novel thermostable P450s with half-lives of inactivation at 57 °C up to 108 times that of the most stable parent. Although they differ by as many as 99 amino acids from any known P450, the stable sequences are catalytically active. Among the novel functions they exhibit is the ability to produce drug metabolites. This chimeric P450 family provides a unique ensemble for biotechnological applications and for studying sequence-stability-function relationships.