Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

Managing sustainable development conflicts: the impact of stakeholders in small-scale hydropower schemes.

The growing importance of the environment and its management has simultaneously emphasized the benefits of hydroelectric power and its environmental costs. In a changing policy climate, giving importance to renewable energy development and environmental protection, conflict potential between stakeholders is considerable. Navigation of conflict determines the scheme constructed, making sustainable hydropower a function of human choice. To meet the needs of practitioners, greater understanding of stakeholder conflict is needed. This paper presents an approach to illustrate the challenges that face small-scale hydropower development as perceived by the stakeholders involved, and how they influence decision-making. Using Gordleton Mill, Hampshire (UK), as an illustrative case, soft systems methodology, a systems modeling approach, was adopted. Through individual interviews, a range of problems were identified and conceptually modeled. Stakeholder bias towards favoring economic appraisal over intangible social and environmental aspects was identified; costs appeared more influential than profit. Conceptual evaluation of the requirements to meet a stakeholder-approved solution suggested a complex linear systems approach, considerably different from the real-life situation. The stakeholders introduced bias to problem definition by transferring self-perceived issues onto the project owner. Application of soft systems methodology caused a shift in project goals away from further investigation towards consideration of project suitability. The challenge of sustainable hydropower is global, with a need to balance environmental, economic, and social concerns. It is clear that in this type of conflict, an individual can significantly influence outcomes; highlighting the need for more structured approaches to deal with stakeholder conflicts in sustainable hydropower development.

Direct measurement of mercury(II) removal from organomercurial lyase (MerB) by tryptophan fluorescence: NmerA domain of coevolved γ-proteobacterial mercuric ion reductase (MerA) is more efficient than MerA catalytic core or glutathione .

Aerobic and facultative bacteria and archaea harboring mer loci exhibit resistance to the toxic effects of Hg(II) and organomercurials [RHg(I)]. In broad spectrum resistance, RHg(I) is converted to less toxic Hg(0) in the cytosol by the sequential action of organomercurial lyase (MerB: RHg(I) → RH + Hg(II)) and mercuric ion reductase (MerA: Hg(II) → Hg(0)) enzymes, requiring transfer of Hg(II) from MerB to MerA. Although previous studies with γ-proteobacterial versions of MerA and a nonphysiological Hg(II)-DTT-MerB complex qualitatively support a pathway for direct transfer between proteins, assessment of the relative efficiencies of Hg(II) transfer to the two different dicysteine motifs in γ-proteobacterial MerA and to competing cellular thiol is lacking. Here we show the intrinsic tryptophan fluorescence of γ-proteobacterial MerB is sensitive to Hg(II) binding and use this to probe the kinetics of Hg(II) removal from MerB by the N-terminal domain (NmerA) and catalytic core C-terminal cysteine pairs of its coevolved MerA and by glutathione (GSH), the major competing cellular thiol in γ-proteobacteria. At physiologically relevant concentrations, reaction with a 10-fold excess of NmerA over HgMerB removes ≥92% Hg(II), while similar extents of reaction require more than 1000-fold excess of GSH. Kinetically, the apparent second-order rate constant for Hg(II) transfer from MerB to NmerA, at (2.3 ± 0.1) × 10(4) M(-1) s(-1), is ∼100-fold greater than that for GSH ((1.2 ± 0.2) × 10(2) M(-1) s(-1)) or the MerA catalytic core (1.2 × 10(2) M(-1) s(-1)), establishing transfer to the metallochaperone-like NmerA domain as the kinetically favored pathway in this coevolved system.

Structural characterization of intramolecular Hg(2+) transfer between flexibly linked domains of mercuric ion reductase.

The enzyme mercuric ion reductase MerA is the central component of bacterial mercury resistance encoded by the mer operon. Many MerA proteins possess metallochaperone-like N-terminal domains (NmerA) that can transfer Hg(2+) to the catalytic core domain (Core) for reduction to Hg(0). These domains are tethered to the homodimeric Core by ~30-residue linkers that are susceptible to proteolysis, the latter of which has prevented characterization of the interactions of NmerA and the Core in the full-length protein. Here, we report purification of homogeneous full-length MerA from the Tn21 mer operon using a fusion protein construct and combine small-angle X-ray scattering and small-angle neutron scattering with molecular dynamics simulation to characterize the structures of full-length wild-type and mutant MerA proteins that mimic the system before and during handoff of Hg(2+) from NmerA to the Core. The radii of gyration, distance distribution functions, and Kratky plots derived from the small-angle X-ray scattering data are consistent with full-length MerA adopting elongated conformations as a result of flexibility in the linkers to the NmerA domains. The scattering profiles are best reproduced using an ensemble of linker conformations. This flexible attachment of NmerA may facilitate fast and efficient removal of Hg(2+) from diverse protein substrates. Using a specific mutant of MerA allowed the formation of a metal-mediated interaction between NmerA and the Core and the determination of the position and relative orientation of NmerA to the Core during Hg(2+) handoff.

A general protocol for the crystallization of membrane proteins for X-ray structural investigation.

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.