Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.

The anionic porphyrin, meso-tetrakis(4-sulfonatophenyl)porphine, is found to tightly bind to an engineered 14-residue peptide, resulting in induced alpha-helix formation when mixed in aqueous solutions. The small porphyrin-peptide dissociation constant (2 muM) observed is related to the energetics of peptide helix formation coupled with electrostatic interactions between the anionic porphyrin and cationic residues in the coiled peptide. Analytical ultracentrifugation measurements indicate the porphyrin-peptide complexes dimerize, probably into a coiled coil, and weakly associate to form even higher order structures.

We are interested in the controlled assembly of photoelectronic materials using peptides as scaffolds and porphyrins as the conducting material. We describe the integration of a peptide-based polymer strategy with the ability of designed basic peptides to bind anionic porphyrins in order to create regulated photoelectronically active biomaterials. We have described our peptide system in earlier work, which demonstrates the ability of a peptide to form filamentous materials made up of self-assembling coiled-coil structures. We have modified this peptide system to include lysine residues appropriately positioned to specifically bind meso-tetrakis(4-sulfonatophenyl)porphine (TPPS(4)), a porphyrin that contains four negatively charged sulfonate groups at neutral pH. We measure the binding of TPPS(4) to our peptide using UV--visible and fluorescence spectroscopies to follow the porphyrin signature. We determine the concomitant acquisition of helical secondary structure in the peptide upon TPPS(4) binding using circular dichroism spectropolarimetry. This binding fosters polymerization of the peptide, as shown by absorbance extinction effects in the peptide CD spectra. The morphologies of the peptide/porphyrin complexes, as imaged by atomic force microscopy, are consistent with the coiled-coil polymers that we had characterized earlier, except that the heights are slightly higher, consistent with porphyrin binding. Evidence for exciton coupling in the copolymers is shown by red-shifting in the UV--visible data, however, the coupling is weak based on a lack of fluorescence quenching in fluorescence experiments.

Evidence is building to support the notion that the porphobilinogen synthase (PBGS 2 ; EC 4.2.1.24) family of enzymes can exist as an equilibrium of quaternary structure isoforms, denoted morpheeins (1-3). Morpheeins comprise an equilibrium ensemble of protein structures wherein a protein monomer can exist in more than one conformation, and each monomer conformation dictates a functionally different quaternary structure of finite multiplicity. Morpheeins have been proposed to provide a structural foundation for allosteric regulation, cooperativity, and hysteresis in some proteins (2). As such, the energetic difference between morpheeins of a given protein must be small. The propensity of PBGS to assume various morpheein structures and the rates of PBGS morpheein interconversion are highly species-dependent. The stable morpheeins of human PBGS are the octamer, found for the wild-type protein, and the hexamer, first seen for the naturally occurring mutation F12L (1). Coexpression of human wild-type PBGS and F12L generates a population of PBGS proteins composed of hetero-octamers and heterohexamers, each of which contains a mixture of Phe 12 -and Leu 12 -containing chains (1). The structure and composition of these hetero-oligomers are stable during storage, but the molecular motions resulting from catalysis favor formation of the octamer with an accompanying disproportionation of Phe 12 -containing chains into the octamer (3).The remaining hexamer has an increased proportion of Leu 12 -containing chains (3). The physical basis for the thermodynamic propensity of Leu 12 -containing chains to form the hexamer remains unclear, but examination of the structure of human PBGS suggests that other single amino acid mutations might affect the folding and assembly of the protein to favor structures other than the octamer. In this study, we report on alterations of two amino acids (Arg 240 and Trp 19 ) that were chosen based on an analysis of the structures and subunit interactions seen in human octameric and hexameric PBGS, for which the assemblies are shown in Fig. 1. Each human PBGS subunit is composed of a 306-amino acid TIM-like ␣␤-barrel and a 24-amino acid N-terminal arm. The conformational difference between the monomer that assembles into the octamer and the monomer that assembles into the hexamer is a dramatic reorientation of the arm with respect to the barrel; for the F12L hexamer, this reorientation occurs at Thr 23 . In both the octameric and hexameric assemblies, two monomers come together to form a dimer with a conserved barrel-barrel interface. The dimer that assembles into the octamer is called a hugging dimer (Fig. 1a), whereas the dimer that assembles into the hexamer is called a detached dimer (Fig. 1b) (1). The difference between these two dimers is the presence or absence of a "hugging" interaction between the N-terminal arm of one subunit and the ␣␤-barrel of the adjacent subunit of the dimer. The "arm-hugging-barrel" interaction of PBGSs from plants, Archaea, and most Bacteria is stabilized by an all...

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. However, many proteins used for therapeutics and biosensors involve beta-sheet proteins such as antibodies. Accordingly, we explored the effect of several highly fluorinated amino acids on beta-sheet stability including (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), (S)-5,5,5',5'-tetrafluoroleucine (Qfl), (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), and (S)-pentafluorophenylalanine (Pff). Nine proteins based on the protein G B1 domain I6A T44A mutant (GB1) with various amino acids at the solvent exposed guest position 53 in the internal strand 4 were synthesized, purified, and investigated by thermal denaturation monitored by circular dichroism spectroscopy. Based on the thermal denaturation data, GB1 stability is affected by the amino acid at the guest position 53. Apparently, introducing fluorine results in more stable GB1 mutants (Pff > Phe, Hfl > Qfl > Leu, Atb > Abu). In particular, GB1 becomes more stable upon introducing fluorines by up to 0.35 kcal x mol(-1) x residue(-1). Overall, these results suggest that fluoro-amino acids may be worthwhile building blocks to explore for stabilizing beta-sheet proteins, which are especially important for biotechnologies such as protein therapeutics and biosensors.

Polyglutamine repeat motifs are known to induce protein aggregation in various neurodegenerative diseases, and flanking sequences can modulate this behavior. It has been proposed that the 17 N-terminal residues (Htt(NT)) of the polyglutamine-containing huntingtin protein accelerate the kinetics of aggregation due to the formation of helix-rich oligomers through helix-pairing interactions. Several hypotheses that explain the role of helical interactions in modulating aggregation have been proposed. These include (1) an increase in the effective concentration of polyglutamine chains (proximity model), (2) the induction of helical structure within the polyglutamine domain itself (transformation model), and/or (3) interdomain interactions between the flanking sequence and the polyglutamine domain (domain cross-talk model). These hypotheses are tested by studying the kinetics of polyglutamine aggregation using a Q25 sequence fused to a well-defined heterotetrameric coiled-coil model system. Using a combined spectroscopic and dye binding approach, it is shown that stable coiled-coil formation strongly inhibits polyglutamine aggregation, suggesting that the proximity and transformation models are insufficient to explain the enhanced aggregation seen in Htt(NT)-polyglutamine constructs. Consistent with other published work, our data support a model in which domain cross-talk prevents formation of a nonspecific aggregated collapsed polyglutamine state, which can act to inhibit conversion to a fibrillar state. Because our model system has a charged to nonpolar residue ratio much higher than that of the Htt(NT) sequence, domain cross-talk is severely weakened, thus favoring the nonspecific aggregation pathway and significantly inhibiting aggregation through a fibrillar pathway.

In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors’ kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.