A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

Biophysical characterization of hit compounds for mechanism-based enzyme activation.

Across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. All of these modes of activation rely on naturally evolved binding sites that trigger global conformational changes. Among the enzymes that are of greatest interest for small molecule activation are the seven sirtuin enzymes, nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Here, we introduce a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds.

Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H.

The phrB gene encoding a putative cold-adapted DNA photolyase was cloned from the bacterial genomic DNA of Colwellia psychrerythraea 34H, a psychrophilic bacterium. Recombinant DNA photolyase, rCpPL, was overexpressed and purified from three different vectors. rCpPL binds its DNA substrate by flipping a cyclobutane pyrimidine dimer (CPD) into its active site and repairs CPD-containing DNA in vitro. rCpPL contains one catalytic flavin adenine dinucleotide (FAD) cofactor, but displays promiscuity in cofactor binding, in which either a flavin mononucleotide (FMN) or a methenyltetrahydrofolate (MTHF) molecule is bound as an antenna molecule and found in sub-stoichiometric amounts. The UV/Vis spectrum of oxidized rCpPL shows that the FADOX absorption maximum is the most red-shifted reported for a PL, suggesting a unique cavity electrostatic environment. Modest FAD vibronic structure suggests that the binding pocket is more flexible than warmer PLs, corroborating the hypothesis that psychrophilic proteins must be highly flexible to function at low temperatures. Fluorescence excitation data show that the freshly purified flavin cofactor is in its fully reduced state (FADH¯). A homology analysis of PL protein structures spanning 70 °C in growth temperature supports the data that the structure of CpPL is quite different from its warmer cousins.

The Missing Electrostatic Interactions Between DNA Substrate and Sulfolobus solfataricus DNA Photolyase: What is the Role of Charged Amino Acids in Thermophilic DNA Binding Proteins?

DNA photolyase can be used to study how a protein with its required cofactor has adapted over a large temperature range. The enzymatic activity and thermodynamics of substrate binding for protein from Sulfolobus solfataricus were directly compared to protein from Escherichia coli. Turnover numbers and catalytic activity were virtually identical, but organic cosolvents may be necessary to maintain activity of the thermophilic protein at higher temperatures. UV-damaged DNA binding to the thermophilic protein is less favorable by ∼2 kJ/mol. The enthalpy of binding is ∼10 kJ/mol less exothermic for the thermophile, but the amount and type of surface area buried upon DNA binding appears to be somewhat similar. The most important finding was observed when ionic strength studies were used to separate binding interactions into electrostatic and nonelectrostatic contributions; DNA binding to the thermophilic protein appears to lack the electrostatic contributions observed with the mesophilic protein.