Benjamin C. Buer

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. Although larger, the fluorinated residues are accommodated within the protein with minimal structural perturbation, because they closely match the shape of the hydrocarbon side chains that they replace. Thus, stability increases seem to be better explained by increases in buried hydrophobic surface area that accompany fluorination than by specific fluorous interactions between fluorinated side chains. This finding is illustrated by the design of a highly fluorinated protein that, by compensating for the larger volume and surface area of the fluorinated side chains, exhibits similar stability to its nonfluorinated counterpart. These structure-based observations should inform efforts to rationally modulate protein function using noncanonical amino acids.

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. Fluorination seems to be a general and effective strategy to enhance the stability of proteins, both soluble and membrane bound, against chemical and thermal denaturation, although retaining structure and biological activity. Most studies have focused on small proteins that can be produced by peptide synthesis as synthesis of large proteins containing specifically fluorinated residues remains challenging. However, the development of various biosynthetic methods for introducing noncanonical amino acids into proteins promises to expand the utility of fluorinated amino acids in protein design.

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.

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. Using the antimicrobial peptide MSI-78 as a model system, we demonstrate that peptide binding to either small unilamellar vesicles (SUVs) or bicelles can readily be detected by simple one-dimensional (19)F NMR experiments with peptides labeled with l-4,4,4-trifluoroethylglycine. The (19)F chemical shift associated with the peptide-membrane complex is sensitive both to the position of the trifluoromethyl reporter group (whether in the hydrophobic face or positively charged face of the amphipathic peptide) and to the curvature of the lipid bilayer (whether the peptide is bound to SUVs or bicelles). (19)F spin echo experiments using the Carr-Purcell-Meiboom-Gill pulse sequence were used to measure the transverse relaxation (T(2)) of the nucleus and thereby examine the local mobility of the MSI-78 analogues bound to bicelles. The fluorine probe positioned in the hydrophobic face of the peptide relaxes at a rate that correlates with the tumbling of the bicelle, suggesting that it is relatively immobile, whereas the probe at the positively charged face relaxes more slowly, indicating this position is much more dynamic. These results are in accord with structural models of MSI-78 bound to lipids and point to the feasibility of using fluorine-labeled peptides to monitor peptide-membrane interactions in living cells.

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. The increase in the free energy of unfolding with increasing fluorine content is associated with increasingly unfavorable entropies of unfolding and correlates well with calculated changes in apolar solvent-accessible surface area. ΔCp° for unfolding is positive for all the proteins and, similarly, correlates with changes in apolar solvent-accessible surface area. ΔH° for unfolding shows no correlation with either fluorine content or changes in apolar solvent-accessible surface area. We conclude that conventional hydrophobic effects adequately explain the enhanced stabilities of most highly fluorinated proteins. The extremely high thermal stability of these proteins results, in part, from their very low per-residue ΔCp°, as has been observed for natural thermostable proteins.

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 1.28 to -1.35 ppm upon binding to lipid bicelles. The largest upfield shifts are associated with the most hydrophobic positions in the peptide. Changes in solvent isotope effects (H(2)O/D(2)O) on (19)F chemical shifts were observed for the peptides that are consistent with the MSI-78 solvent-inaccessible hydrophobic core upon binding bicelles. Transverse relaxation measurements of the (19)F nucleus, using the Carr-Purcell-Meiboom-Gill pulse sequence, were used to examine changes in the local mobility of MSI-78 that occur upon binding to the lipid bilayer. Positions in the hydrophobic core of peptide-membrane complex show the greatest decrease in mobility upon binding of the lipid bilayer, whereas residues that interact with lipid headgroups are more mobile. The most mobile positions are at the N- and C-termini of the peptide. These results provide support for the proposed mechanism of membrane disruption by MSI-78 and reveal new details about the dynamic changes that accompany membrane binding.

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

Fluorine (19F) 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 19F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI‐78 results in a sharp 19F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI‐78 provides a sensitive tool to study binding through 19F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in‐cell investigations. Copyright

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. The structures also identify a water channel leading to the active site that could facilitate the entry of protons required in the reaction. NMR studies examining 1-[13C]-octanal binding to cADO indicate that the enzyme binds the aldehyde form rather than the hydrated form. Lastly, the fortuitous cocrystallization of the metal-free form of the protein with aldehyde bound has revealed protein conformation changes that are involved in binding iron.

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. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.

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, Alzheimers 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. Such problems highlight the potential utility of a label-free high throughput screening (HTS) strategy. In this work, we describe a label-free SIRT1 assay suitable for HTS based on segmented flow-electrospray ionization-mass spectrometry (ESI-MS). In the assay, 0.5 μM SIRT1 was incubated with 20 μM acetylated 21-amino acid peptide, which acts as substrate for the protein. A stable-isotope labeled product peptide was added to the assay mixture as an internal standard after reaction quenching. The resulting samples are formatted into 100 nL droplets segmented by perfluorodecalin and then infused at 0.8 samples/s into an ESI-MS. To enable direct ESI-MS analysis, 11 μM SIRT1 was dialyzed into a 200 μM ammonium formate (pH 8.0) buffer prior to use in the assay. This buffer was demonstrated to minimally affect enzyme kinetics and yet be compatible with ESI-MS. The assay conditions were optimized through enzyme kinetic study, and tested by screening an 80-compound library. The assay Z-factor was 0.7. Four inhibitors and no activators were detected from the library.