Biomedical subjects

Trevor J Rutherford

Publications and source records attributed to Trevor J Rutherford.

5 recordsLinked to original sources

PubMed2006

Solution structure of p53 core domain: structural basis for its instability.

The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using state-of-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.

Crystallography↗

PubMed2005

Ultra-fast barrier-limited folding in the peripheral subunit-binding domain family.

We have determined the solution structures, equilibrium properties and ultra-fast folding kinetics for three bacterial homologues of the peripheral subunit-binding domain (PSBD) family. The mesophilic homologue, BBL, was less stable than the thermophilic and hyper-thermophilic variants (E3BD and POB, respectively). The broad unfolding transitions of each PSBD, when probed by different techniques, were essentially superimposable, consistent with co-operative denaturation. Temperature-jump and continuous-flow fluorescence methods were used to measure the folding kinetics for E3BD, POB and BBL. E3BD folded fairly rapidly at 298K (folding half-time approximately 25 micros) and BBL and POB folded even faster (folding half-times approximately 3-5 micros). The variations in equilibrium and kinetic behaviour observed for the PSBD family resembles that of the homeodomain family, where the folding pattern changes from apparent two-state transitions to multi-state kinetics as the denatured state becomes more structured. The faster folding of POB may be a consequence of its higher propensity to form helical structure in the region corresponding to the folding nucleus of E3BD. The ultra-fast folding of BBL appears to be a consequence of residual structure in the denatured ensemble, as with engrailed homeodomain. We discuss issues concerning "one-state", downhill folding, and find no evidence for, and strong evidence against, it occurring in these PSBDs. The shorter construct used previously for BBL was destabilized significantly and the stability further perturbed by the introduction of fluorescent probes. Thermal titrations for 11 side-chains scattered around the protein, when probed by (13)C-NMR experiments, could be fit globally to a common co-operative transition.

Amino Acid Sequence↗

PubMed2004

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding.

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.

Alanine↗

PubMed2004

Hydrophobic core fluidity of homologous protein domains: relation of side-chain dynamics to core composition and packing.

The side-chain dynamics of methyl groups in two structurally related proteins from the fibronectin type III (fnIII) superfamily, the third fnIII domain from human tenascin (TNfn3) and the tenth fnIII domain from human fibronectin (FNfn10), have been studied by NMR spectroscopy. Side-chain order parameters reveal that the hydrophobic cores of the two proteins have substantially different mobilities. The core of TNfn3 is very dynamic, with exceptionally low order parameters for the most deeply buried residues, while that of FNfn10 is more like those of other proteins which have been studied with this technique, having a relatively rigid core with uniformly distributed dynamics. The unusually dynamic core of TNfn3 appears to be related to its amino acid composition, which makes it more fluid-like. A further explanation for the mobility of the TNfn3 core may be found in the negative correlation between the order parameter and excess packing volume, which shows that the core of TNfn3 is less densely packed and consequently has lower methyl order parameters for its buried residues. Rotameric transitions, presumably facilitated by the lower packing density, appear to make an important contribution to lowering the order parameters, and have been probed by measuring three-bond scalar couplings. Overall, although backbone dynamics is generally similar for proteins with the same topology on a fast time scale (picoseconds to nanoseconds), this study shows that a single fold can accommodate a wide variation in the dynamic properties of its core.

Amino Acid Sequence↗

PubMed2002

Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.

Computer Simulation↗