Biomedical subjects

Houbi Nguyen

Publications and source records attributed to Houbi Nguyen.

6 recordsLinked to original sources

PubMed2006

Structure-function-folding relationship in a WW domain.

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and/or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I' beta-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.

Amino Acid Sequence↗

PubMed2006

Beta-sheet folding mechanisms from perturbation energetics.

Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional beta-sheets, consistent with evolutionary robustness.

Animals↗

PubMed2005

Engineering a beta-sheet protein toward the folding speed limit.

Recent experimental studies have shown that alpha-helical proteins can approach the folding "speed limit", where folding switches from an activated to a downhill process in free energy. beta-sheet proteins are generally thought to fold more slowly than helix bundles. However, based on studies of hairpins, folding should still be able to approach the microsecond time scale. Here we demonstrate how the hPin1 WW domain, a triple-stranded beta-sheet protein with a sharp thermodynamic melting transition, can be engineered toward the folding "speed limit" without a significant loss in thermal denaturation cooperativity.

Amino Acid Sequence↗

PubMed2004

Context-dependent contributions of backbone hydrogen bonding to beta-sheet folding energetics.

Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small beta-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a beta-sheet protein.

Amides↗

PubMed2003

Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation.

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded beta-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28-37 residues), the availability of a quantitative reaction coordinate (phi(T)), the fast folding time scale (10s of micros), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory.

Amino Acid Sequence↗

PubMed2002

Absolute comparison of simulated and experimental protein-folding dynamics.

Protein folding is difficult to simulate with classical molecular dynamics. Secondary structure motifs such as alpha-helices and beta-hairpins can form in 0.1-10 micros (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- micro s 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 micros) 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.

Amino Acid Sequence↗