De novo design of a non-local β-sheet protein with high stability and accuracy.

β-sheet proteins carry out critical functions in biology, and hence are attractive scaffolds for computational protein design. Despite this potential, de novo design of all-β-sheet proteins from first principles lags far behind the design of all-α or mixed-αβ domains owing to their non-local nature and the tendency of exposed β-strand edges to aggregate. Through study of loops connecting unpaired β-strands (β-arches), we have identified a series of structural relationships between loop geometry, side chain directionality and β-strand length that arise from hydrogen bonding and packing constraints on regular β-sheet structures.

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure.

Automating human intuition for protein design.

In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved.

A Pareto-optimal refinement method for protein design scaffolds.

Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or "scaffolds", are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein-protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints.

Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction.

The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS).

Thermodynamics and kinetics of the hairpin ribozyme from atomistic folding/unfolding simulations.

We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a Monte Carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure.

Two distinct binding modes of a protein cofactor with its target RNA.

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure.

Prion protein NMR structures of cats, dogs, pigs, and sheep.

The NMR structures of the recombinant cellular form of the prion proteins (PrPC) of the cat (Felis catus), dog (Canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented. In all of these species, PrPC consists of an N-terminal flexibly extended tail with approximately 100 amino acid residues and a C-terminal globular domain of approximately 100 residues with three alpha-helices and a short antiparallel beta-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrPC structures, there are local differences between the globular domains of the different species.

All-atom Monte Carlo simulation of GCAA RNA folding.

We report a detailed all-atom simulation of the folding of the GCAA RNA tetraloop. The GCAA tetraloop motif is a very common and thermodynamically stable secondary structure in natural RNAs. We use our simulation methods to study the folding behavior of a 12-base GCAA tetraloop structure with a four-base helix adjacent to the tetraloop proper.

Amino acid sequence of the Felis catus prion protein.

A new determination of the house cat (Felis catus) prion protein gene sequence (fPrnp), which has so far been subject of controversy, is reported. The newly determined fPrnp sequence is similar to dog (Canis familiaris) and mink (Mustela putorius) Prnp, but differs significantly from both fPrnp sequences that were previously deposited in the GenBank. Comparison of the canine and feline prion protein sequences suggests a set of amino acid replacements relative to bovine PrP that might relate to the observed different susceptibilities of the two species to TSE infection by ingestion of BSE-infected beef.

Effects of chronic stress on hippocampal long-term potentiation.

Chronic stress causes atrophy of the apical dendrites of CA3 pyramidal neurons and deficits in spatial memory. We investigated the effects of chronic stress on hippocampal physiology and long-term potentiation (LTP) in the CA3 and dentate gyrus (DG). Rats were subjected to chronic (21 days, 6 h/day) restraint stress and tested for LTP 48 h following the last stress episode.