Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin.

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems

Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu and His Residues

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster be-tween +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformation-al change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region

Electron and Proton Transfers Modulate DNA Binding by the Transcription Regulator RsrR

The [Fe2S2]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed (Out) and buried (In) states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the Out and In states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the In state due to a change in its pKa caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes. Our findings illustrate a novel function of protonation resulting from electron transfer

X-ray crystallographic studies of metalloproteins.

International audienceMany proteins require metals for their physiological function. In combination with spectroscopic characterizations, X-ray crystallography is a very powerful method to correlate the function of protein-bound metal sites with their structure. Due to their special X-ray scattering properties, specific metals may be located in metalloprotein structures and eventually used for phasing the diffracted X-rays by the method of Multi-wavelength Anomalous Dispersion (MAD). How this is done is the principle subject of this chapter. Attention is also given to the crystallographic characterization of different oxidation states of redox active metals and to the complication of structural changes that may be induced by X-ray irradiation of protein crystals

Structural Foundations for O2 Sensitivity and O2 Tolerance in [NiFe]-Hydrogenases

International audienceNature has evolved three different ways of metabolizing hydrogen, represented by the anaerobic [Fe]-, [FeFe]- and [NiFe]-hydrogenases. Structural and functional studies of these enzymes have unveiled the unusual composition of their active sites and characterized their catalytic mechanisms. From a biotechnological viewpoint, the most interesting hydrogenases are those that contain a [NiFe] moiety in their active sites. Some of these enzymes are O2-resistant and can rapidly reductively recover from oxygen exposure whereas others are O2-tolerant and can oxidize H2 even at atmospheric oxygen levels. O2-resistant [NiFeSe]-hydrogenases have one of the Cys ligands of the active site replaced by a SeCys and do not display the hard-to-reactivate “unready” state provoked by O2. The reasons for this property might be related to the formation of O2-derived Se-O bonds, which are weaker than S-O bonds and, consequently, easier to break upon reduction. Conversely, membrane-bound O2-tolerant hydrogenases have an unusual proximal (relative to the active site) [Fe4S3] cluster coordinated by six Cys ligands. This cluster can rapidly send two successive electrons to the active site helping to reduce oxygen to water there. Some microorganisms posses more than one hydrogenase and use them in different ways. For instance, there are three well-characterized [NiFe]-hydrogenases in the model bacterium Escherichia coli. They are highly regulated and each one plays a specific role: microaerobic/anaerobic H2 uptake, anaerobic H2 evolution and, protection from O2-induced damage, respectively. These enzymes are discussed in connection with the metabolic changes E. coli undergoes during its transit through the intestinal tract of the host. O2-tolerant hydrogenases have been used to build bio-fuel cells that can function under air. Also, O2-resistant [NiFeSe]-hydrogenases have been attached to TiO2 particules for H2 production from solar energy. Hydrogenase active sites have also served as a source of inspiration for the synthesis of organometallic catalysts