BackgroundPsychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.ResultsWe examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.ConclusionsThese findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth β-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of β-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 105 sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C-termini is formed, folding is 23 faster, unfolding is 20003 slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m-value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink-induced perturbations in kinetics are useful for mapping the protein folding pathway.

Programs for splitting protein design expressions are available at www.bioinfo.rpi.edu/tools/piecemeal.html CONTACT: bystrc@rpi.edu Supplementary information: Supplementary data are available at Bioinformatics online.