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Jody M Mason

Publications and source records attributed to Jody M Mason.

9 recordsLinked to original sources
PubMed2007

A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability.

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such proteins, but the results to date have been of mixed success. Here, we present a robust method involving the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally, selection at physiological temperatures, to generate an enzyme with improved stability. Three cycles of directed evolution comprising of random mutagenesis, DNA shuffling, and selection at 37 degrees C were used, using the bacterial enzyme TEM-1 beta-lactamase as a model protein to yield deletion mutants with in vivo ampicillin resistance levels comparable to wild-type (wt) enzyme. Kinetic studies demonstrate the selected mutant to have a significantly improved thermostability relative to its wt counterpart. Elongation of this mutant to the full-length gene resulted in a beta-lactamase variant with dramatically increased thermostability. This technique was so fruitful that the evolved enzyme retained its maximum catalytic activity even 20 degrees C above its wt parent protein optimum. Thus, structural perturbation by terminal truncation and subsequent compensation by directed evolution at physiological temperatures is a fast, efficient, and highly effective way to improve the thermostability of proteins without the need for selecting at elevated temperatures.

Amino Acid Sequence↗
PubMed2007

Considerations in the design and optimization of coiled coil structures.

Coiled coil motifs are, despite their apparent simplicity, highly specific, and play a significant role in the understanding of tertiary structure and its formation. The most commonly observed of the coiled coils, the parallel dimeric, is yet to be fully characterized for this structural class in general. Nonetheless, strict rules have emerged for the necessity of specific types of amino acids at specific positions. In this chapter, we discuss this system in light of existing coiled coil structures and in applying rules to coiled coils that are to be designed or optimized. Understanding and expanding on these rules is crucial in using these motifs, which play key roles in virtually every cellular process, to act as drug-delivery agents by sequestering other proteins that are not behaving natively or that have been upregulated (for example, by binding to coiled coil domains implicated in oncogenesis). The roles of the a and d "hydrophobic" core positions and the e and g "electrostatic" edge positions in directing oligomerization and pairing specificity are discussed. Also discussed is the role of these positions in concert with the b, c, and f positions in maintaining alpha-helical propensity, helix solubility, and dimer stability.

Amino Acid Motifs↗
PubMed2006

N-Methylated peptide inhibitors of beta-amyloid aggregation and toxicity. Optimization of the inhibitor structure.

The key pathogenic event in the onset of Alzheimer's disease (AD) is believed to be the aggregation of the beta-amyloid (Abeta) peptide into toxic oligomers. Molecules that interfere with this process may therefore act as therapeutic agents for the treatment of AD. N-Methylated peptides (meptides) are a general class of peptide aggregation inhibitors that act by binding to one face of the aggregating peptide but are unable to hydrogen bond on the other face, because of the N-methyl group replacing a backbone NH group. Here, we optimize the structure of meptide inhibitors of Abeta aggregation, starting with the KLVFF sequence that is known to bind to Abeta. We varied the meptide length, N-methylation sites, acetylation, and amidation of the N and C termini, side-chain identity, and chirality, via five compound libraries. Inhibitor activity was tested by thioflavin T binding, affinity chromatography, electron microscopy, and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide toxicity assay. We found that inhibitors should have all d chirality, have a free N terminus but an amidated C terminus, and have large, branched hydrophobic side chains at positions 1-4, while the side chain at position 5 was less important. A single N-methyl group was necessary and sufficient. The most active compound, d-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH(2), was more active than all previously reported peptide inhibitors. Its related non-N-methylated analogues were insoluble and toxic.

Amino Acid Sequence↗
PubMed2006

Semirational design of Jun-Fos coiled coils with increased affinity: Universal implications for leucine zipper prediction and design.

Activator protein-1 (AP-1) is a crucial transcription factor implicated in numerous cancers. For this reason, nine homologues of the AP-1 leucine zipper region have been characterized: Fos (c-Fos, FosB, Fra1, and Fra2), Jun (c-Jun, JunB, and JunD), and semirational library-designed winning peptides FosW and JunW. The latter two were designed to specifically target c-Fos or c-Jun. They have been identified by using protein-fragment complementation assays combined with growth competition. This assay removes nonspecific, unstable, and protease susceptible library members from the pool, leaving winners with excellent drug potential. Thermal melts of all 45 possible dimeric interactions have been surveyed, with the FosW-c-Jun complex displaying a melting temperature (T(m)) of 63 degrees C, compared to only 16 degrees C for wild-type c-Fos-c-Jun interaction. This impressive 70,000-fold K(D) decrease is largely due to optimized core packing, alpha-helical propensity, and electrostatics. Contrastingly, due to a poor c-Fos core, c-Fos-JunW dimerizes with lower affinity. However the T(m) far exceeds wild-type c-Fos-c-Jun and averaged JunW and c-Fos, indicating a preference over either homodimer. Finally, and with wider implications, we have compiled a method for predicting interaction of parallel, dimeric coiled coils, using our T(m) data as a training set, and applying it to 59 bZIP proteins previously reported. Our algorithm, unlike others to date, accounts for helix propensity, which is found to be integral in coiled coil stability. Indeed, in applying the algorithm to these 59(2) bZIP interactions, we were able to correctly identify 92% of all strong interactions and 92% of all noninteracting pairs.

Amino Acid Sequence↗
PubMed2006

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding.

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.

Bacteriorhodopsins↗
PubMed2005

Low energy pathways and non-native interactions: the influence of artificial disulfide bridges on the mechanism of folding.

Four versions of a beta-sheet protein (CD2.d1) have been made, each with a single artificial disulfide bond inserted into hairpin structures. Folding kinetics of reduced and oxidized forms shows bridge position strongly influences its effect on the folding reaction. Bridging residues 58 and 62 does not affect the rapidly formed intermediate (I) or rate-limiting transition (t) state, whereas bridging 33 and 38, or 31 and 41, lowers the t-state energy, with the latter having the stronger influence. Bridging residues 79 and 90 stabilizes both I- and t-states. To assess additivity in the energetic effects of these bridges, four double-bridge variants have also been made. All show precise additivity of overall stability, with two showing additivity when ground states and the rate-limiting t-state are assessed, i.e. no measurable change in the folding mechanism occurs. However, combining 31-41 and 79-90 bridges produces a molecule that folds through a different pathway, with a much more stable intermediate than expected and a much higher t-state barrier. This is explained by the artificial introduction of stabilizing, non-native contacts in the I-state. More surprisingly, for another double-bridge version (58-62 and 79-90) both I- and t-states are less stable than expected, showing that conformational constraints introduced by the two bridges prevent formation of non-native contacts that would otherwise stabilize the I- and t-states, thereby lowering the energy of the folding landscape in the wild-type (unbridged) molecule. We conclude that the lowest energy path for folding has I- and t-state structures that are stabilized by non-native interactions.

Animals↗
PubMed2003

Design strategies for anti-amyloid agents.

Numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptides clump together in the brain or body to form toxic soluble oligomers and/or insoluble fibres. An attractive strategy to develop therapies for these diseases is therefore to inhibit or reverse protein/peptide aggregation. A diverse range of small organic ligands have been found to act as aggregation inhibitors. Alternatively, the wild-type peptide can be derivatised so that it still binds to the amyloid target, but prevents further aggregation. This can be achieved by adding a bulky group or charged amino acid to either end of the peptide, or by incorporating proline residues or N-methylated amide groups.

Amyloid beta-Peptides↗
PubMed2002

The influence of intramolecular bridges on the dynamics of a protein folding reaction.

Thirteen versions of a beta-sheet protein have been constructed, each with a single, surface-exposed disulfide bridge. A comparison of folding kinetics, in oxidizing and reducing conditions, is used to elucidate the order in which beta-strands become associated during the folding process and, hence, the relationship between topology and folding dynamics. In common with the wild-type molecule, all the proteins fold through a two-step (three state) mechanism with a rapidly formed intermediate which slowly converts to the native state. In a majority of cases, the bridge is seen to stabilize the folded state, and for five of the modified proteins, the additional stability is greater than 3 kcal/mol. Surprisingly, cross-links which connect beta-strands which are distant in sequence predominantly stabilize the rapidly formed intermediate state, suggesting that these strand-strand interactions occur in the initial stages of folding. Cross-links which stabilize local hairpins have their major influence on the second, rate-determining step leading to significant enhancements in the folding rate. We find that enhancement of the folding rate in the second, rate-limiting step is correlated with a reduction in contact order in the same way as in naturally occurring proteins of different folds. The large increases in native-state stability resulting from the insertion of disulfide bridges on the surface of beta-sheet structures have implications for enhancing the robustness of proteins by molecular engineering.

Animals↗