Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.

Sum frequency generation (SFG) vibrational spectroscopy has been applied to investigate peptide immobilization to a polymer surface as a function of time and peptide conformation. Surface immobilization of biological molecules is important in many applications such as biosensors, antimicrobial materials, bio-based fuel cells, nanofabrication, and multi-functional materials. Using C-terminus cysteine modified cecropin P1 (CP1c) as a model, we investigated the time-dependent immobilization behavior in situ in real time. In addition, potassium phosphate buffer (PB) and mixtures of PB and trifluoroethanol were utilized to examine the effect of peptide secondary structure on CP1c immobilization to polystyrene maleimide (PS-MA). The orientation of immobilized CP1c on PS-MA was determined using polarized SFG spectra. It was found that the peptide solution concentration, solvent composition, and assembly state (monomer vs. dimer) prior to immobilization all influence the orientation of CP1c on a PS-MA surface. The detailed relationship between interfacial peptide orientation and these immobilization conditions is discussed.

Oligomers based on an o-phenylene ethynylene (oPE) backbone with polar substituents have been synthesized using Sonogashira methods. Folding of these extremely short oligomers was confirmed via 1D and 2D (NOESY) NMR methods. Utilizing electron-rich and electron-poor phenylene building blocks, variations of these oPE oligomers have been synthesized to determine the folded stability of pi-rich vs pi-poor vs pi-rich-pi-poor systems. Slight variations in temperature offer a route, aside from solvent denaturation, to probe the stability of the folded structure. This is the first report of an NMR solution characterization of folding for a PE backbone without hydrogen bonds.

Cation-interactions between aromatic amino acids and the positively charged residues lysine and arginine have been proposed to play an important role in stabilizing protein structure. We have used a peptide that adopts a coiled coil structure as a model system to evaluate the energetic contribution of cation-interactions to protein folding. Peptides were designed in which phenylalanine, tyrosine, and tryptophan were placed at a solvent-exposed position of the helix, one turn removed from an arginine residue that could provide a favorable cation-interaction. Only the arginine-phenylalanine pairing provided significant stabilization of the peptide structure and it appears that hydrophobic packing, rather than the cation-effect, is more likely to be responsible for the stability of this peptide. We conclude that any stabilizing effect of cation-interactions in these peptides is much smaller than that predicted from computational studies.Keywords: de novo designed proteins; protein stability; ␣ helix; cationSupplemental material: see www.proteinscience.orgThe cation-effect arises from favorable electrostatic interactions between the electron-rich system of an aromatic molecule and a positively charged species such as a metal ion or quaternary amine (Ma and Dougherty 1997;Waters 2002). Interactions between aromatic amino acid residues and positively charged side chains, attributed to the cation-effect, are commonly observed in proteins (Burley and Petsko 1985;Dougherty 1996). They have attracted interest as they represent a novel type of noncovalent interaction between hydrophobic and hydrophilic side chains that has the potential to confer both stability and specificity in protein folding. Similar interactions also appear to be important in the binding of positively charged substrates in enzymes such as acetylcholine esterase and trimethylamine dehydrogenase, where the active sites contain numerous aromatic residues but no negatively charged residues that could formally neutralize the positive charge of the substrate (Wilson et al. 1995;Scrutton and Raine 1996;Beene et al. 2002;Zacharias and Dougherty 2002).The strength of the cation-interaction has been investigated in theoretical studies, and experimentally in gasphase experiments, in small molecule host-guest model systems in solution, and in proteins (Sunner et al. 1981;Deakyne and Meotner 1985;Dougherty 1996;Ma and Dougherty 1997;Gallivan and Dougherty 2000). A concise and critical summary of much of this previous work is provided by Kallenbach (Shi et al. 2002b). In particular, theoretical studies by Gallivan and Dougherty concluded that cation-interactions in proteins may attain a strength of up to 4 kcal/mole and are potentially more stabilizing than salt bridges (Gallivan andDougherty 1999, 2000). However, experimentally the magnitude of cation-effects in proteins remains poorly defined, because of the difficulty of dissecting out this energetic term from other noncovalent interactions, in particular hydrophobic effects, which contribute to protein ...

We have previously reported the synthesis of short o-phenylene ethynylene oligomers with polar triethylene glycol side chains which adopt a helical conformation in solution with three residues per turn. Two new oligomers have been synthesized, a hexamer and a nonamer, incorporating a repeated triad motif of polar-nonpolar-polar sidechains in order to create a hydrophobic stripe in the folded conformation which we report here for the first time. Helical folding in solution was observed and, unlike the previously-reported oligomers, these new oligomers are ordered solids at room temperature. Although these oligomers were designed to assemble into helical bundle-like structures, no evidence for a quaternary-like structure was found. The difference in polarity between alkyl and triethylene glycol side chains is likely not strong enough to induce self-association of folded helices, especially since the molecules are not water soluble where the driving force for association of the nonpolar stripe would be larger. We expect that more polar side chains, granting water solubility, represent an important target for future research.

Electrostatic nanoassemblies were employed to identify bacterial growth conditions. They comprise a cationic conjugated oligoelectrolyte and fluorescein-tagged ssDNA and were optimized with a hybrid, computational neural network model. The photoluminescence spectra contained the oligomer and sensitized fluorescein emission. The spectra changed depending on the growth history of the bacteria introduced (see figure).

This paper describes the characterization of solvent induced folding behavior for non-polar (NP) alkoxy substituted ortho-phenylene ethynylene (o-PE) oligomers. Oligomers of lengths up to nine units have been shown to adopt helical conformations in heptane by NMR and CD spectroscopy, while chloroform promotes extended conformations. Surprisingly, the molar ellipticity values found in heptane for these oligomers are very small compared to other literature values of meta-phenylene ethynylene (m-PE) folded systems; however, comparable molar ellipticity values were found for a closed macrocyclic o-PE suggesting the weak ellipticity is a molecular-feature rather than a quality of folding indicator.

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