Experimental approaches to kinetics of gas diffusion in hydrogenase

Hydrogenases, which catalyze H2 to H+ conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these

Validation of crystallographic models containing TLS or other descriptions of anisotropy

Guidelines and specific tests for validating macromolecular crystal structures that include TLS models are introduced. Validation may used to troubleshoot problems during refinement, to confirm the internal consistency of the model as part of deposition into the Protein Data Bank or to assess the plausibility of interpretating the boundary between two TLS groups as indicating a hinge point between structural domains

Towards a resilience management guideline—Cities as a starting point for societal resilience

Unexpected crises and risks affect the urban population. Critical infrastructure dependency, climate change and social dynamics have captured the attention of city decision makers across different disciplines, sectors, and scales. Addressing these challenges mandates an increase in resilience. This article presents the development of the novel European Resilience Management Guideline (ERMG) developed by the European H2020 Smart Mature Resilience (SMR) project. It encompasses five supporting tools for city resilience. The purpose of this article is threefold. First, it describes the extensive co-creation methods used to establish, validate and test the five ERMG tools as collaborations among seven city stakeholders and researchers in Europe. Second, it explains concisely the features of each tool and its use and applicability in the city resilience building process. Third, it shows how EMRG supports strategic management in encouraging the visibility of risk dependencies, identifying vicious loops and potential cascading effects, and promoting collaboration between stakeholders to share resources. The article concludes with a discussion of SMR standardization activities to support the transfer of these research results to wider audiences. It covers guidance on local resilience planning and supporting efforts in building and operationalizing resilience at the city level