A Label-free Sirtuin 1 Assay based on Droplet-Electrospray Ionization Mass Spectrometry.

Sirtuin 1(SIRT1) is a NAD-dependent deacetylase which has been implicated in age-related diseases such as cancer, Alzheimer's disease, type 2 diabetes, and vascular diseases. SIRT1 modulators are of interest for their potential therapeutic use and potential as chemical probes to study the role of SIRT1. Fluorescence-based assays used to identify SIRT1 activators have been shown to have artifacts related to the fluorophore substrates used in the assays.

Insights into substrate and metal binding from the crystal structure of cyanobacterial aldehyde deformylating oxygenase with substrate bound.

The nonheme diiron enzyme cyanobacterial aldehyde deformylating oxygenase, cADO, catalyzes the highly unusual deformylation of aliphatic aldehydes to alkanes and formate. We have determined crystal structures for the enzyme with a long-chain water-soluble aldehyde and medium-chain carboxylic acid bound to the active site. These structures delineate a hydrophobic channel that connects the solvent with the deeply buried active site and reveal a mode of substrate binding that is different from previously determined structures with long-chain fatty acids bound.

Design, synthesis, and study of fluorinated proteins.

Highly fluorinated analogs of hydrophobic amino acids have proven to be generally effective in increasing the thermodynamic stability of proteins. These non-proteogenic amino acids can be incorporated into both α-helix and β-sheet structural motifs and generally enhance protein stability towards unfolding by heat and chemical denaturants, and retard their degradation by proteases. Recent detailed structural and thermodynamic studies have demonstrated that the increase in buried hydrophobic surface area that accompanies fluorination is primarily responsible for the stabilizing properties of fluorinated side chains.

Perfluoro-tert-butyl-homoserine as a sensitive 19F NMR reporter for peptide-membrane interactions in solution.

Fluorine ((19)F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, L-O-(perfluoro-t-butyl)-homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used (19)F biomolecular NMR probes.

Comparison of the structures and stabilities of coiled-coil proteins containing hexafluoroleucine and t-butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side-chains.

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4-helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t-butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein.

Influence of fluorination on the thermodynamics of protein folding.

The introduction of highly fluorinated analogues of hydrophobic amino acid residues into proteins has proved an effective and general strategy for increasing protein stability toward both chemical denaturants and heat. However, the thermodynamic basis for this stabilizing effect, whether enthalpic or entropic in nature, has not been extensively investigated. Here we describe studies in which the values of ΔH°, ΔS°, and ΔCp° have been determined for the unfolding of a series of 12 small, de novo-designed proteins in which the hydrophobic core is packed with various combinations of fluorinated and non-fluorinated amino acid residues.

Structural basis for the enhanced stability of highly fluorinated proteins.

Noncanonical amino acids have proved extremely useful for modifying the properties of proteins. Among them, extensively fluorinated (fluorous) amino acids seem particularly effective in increasing protein stability; however, in the absence of structural data, the basis of this stabilizing effect remains poorly understood. To address this problem, we solved X-ray structures for three small proteins with hydrophobic cores that are packed with either fluorocarbon or hydrocarbon side chains and compared their stabilities.

Fluorine: a new element in protein design.

Fluorocarbons are quintessentially man-made molecules, fluorine being all but absent from biology. Perfluorinated molecules exhibit novel physicochemical properties that include extreme chemical inertness, thermal stability, and an unusual propensity for phase segregation. The question we and others have sought to answer is to what extent can these properties be engineered into proteins? Here, we review recent studies in which proteins have been designed that incorporate highly fluorinated analogs of hydrophobic amino acids with the aim of creating proteins with novel chemical and biological properties.

Using fluorine nuclear magnetic resonance to probe changes in the structure and dynamics of membrane-active peptides interacting with lipid bilayers.

The antimicrobial peptide MSI-78 serves as a model system for studying interactions of bioactive peptides with membranes. Using a series of MSI-78 peptides that incorporate l-4,4,4-trifluoroethylglycine, a small and sensitive (19)F nuclear magnetic resonance probe, we investigated how the local structure and dynamics of the peptide change when it binds to the lipid bilayer. The fluorinated MSI-78 analogues exhibited position-specific changes in (19)F chemical shift ranging from 1.

Using fluorine nuclear magnetic resonance to probe the interaction of membrane-active peptides with the lipid bilayer.

A variety of biologically active peptides exert their function through direct interactions with the lipid membrane of the cell. These surface interactions are generally transient and highly dynamic, making them hard to study. Here we have examined the feasibility of using solution phase (19)F nuclear magnetic resonance (NMR) to study peptide-membrane interactions.

Engineering protein stability and specificity using fluorous amino acids: the importance of packing effects.

The incorporation of extensively fluorinated, or fluorous, analogues of hydrophobic amino acids into proteins potentially provides the opportunity to modulate the physicochemical properties of proteins in a predictable manner. On the basis of the properties of small fluorocarbon molecules, extensively fluorinated proteins should be both more thermodynamically stable and self-segregate through "fluorous" interactions between fluorinated amino acids. We have examined the effects of introducing the fluorous leucine analogue l-5,5,5,5',5',5',-hexafluoroleucine (hFLeu) at various positions within the hydrophobic core of a de novo-designed four-alpha-helix bundle protein, alpha(4).

Fluorine--a new element in the design of membrane-active peptides.

Antimicrobial peptides (also known as genetically encoded peptide antibiotics) are a diverse class of short cationic amphipathic polypeptides that exhibit a broad-spectrum of antimicrobial activities by selectively disrupting the bacterial cell membrane. In this review article, we present the use of fluorinated amino acids in the design of antimicrobial peptides and other membrane-active peptides.