Compatibility of the conformationally rigid CF3-Bpg side chain with the hydrophobic coiled-coil interface

3-(Trifluoromethyl)bicyclopent-[1.1.1]-1-yl glycine (CF₃-Bpg) has previously been established as a useful ¹⁹F NMR label to analyse the structures of oligomeric membrane-active peptides or transmembrane segments. To systematically examine the effect of side chain volume, conformational rigidity, and hydrophobicity of CF₃-Bpg in polypeptide environments the amino acid was incorporated into an established coiled-coil based screening system. A single substitution of either valine (position a16) or leucine (position d19) within the hydrophobic core of the heteromeric coiled coil has practically no effect on its structure. Despite its comparatively high hydrophobicity, however, the stiff and bulky side chain of CF₃-Bpg is not so well accommodated by the hydrophobic core as it leads to a more pronounced destabilization than observed for other, more polar fluorinated amino acids which carry more flexible side chains. CF₃-Bpg is therefore a useful ¹⁹F NMR label, though not for monitoring the stability of such helix–helix interactions.     

Solution structure and dynamics of a designed hydrophobic core variant of ubiquitin.

BACKGROUND: The recent merger of computation and protein design has resulted in a burst of success in the generation of novel proteins with native-like properties. A critical component of this coupling between theory and experiment is a detailed analysis of the structures and stabilities of designed proteins to assess and improve the accuracy of design algorithms. RESULTS: Here we report the solution structure of a hydrophobic core variant of ubiquitin, referred to as 1D7, which was designed with the core-repacking algorithm ROC. As a measure of conformational specificity, we also present amide exchange protection factors and backbone and sidechain dynamics. The results indicate that 1D7 is similar to wild-type (WT) ubiquitin in backbone structure and degree of conformational specificity. We also observe a good correlation between experimentally determined sidechain structures and those predicted by ROC. However, evaluation of the core sidechain conformations indicates that, in general, 1D7 has more sidechains in less statistically favorable conformations than WT. CONCLUSIONS: Our results provide an explanation for the lower stability of 1D7 compared to WT, and suggest modifications to design algorithms that may improve the accuracy with which structure and stability are predicted. The results also demonstrate that core packing can affect conformational flexibility in subtle ways that are likely to be important for the design of function and protein-ligand interactions.     

The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N‐domain of cardiac troponin C (F77W‐cNTnC) to study the domain orientation of troponin C in muscle fibers using solid‐state NMR. In our previous study, we determined the NMR structure of calcium‐saturated mutant F77W‐V82A‐cNTnC in the presence of 19% 2,2,2‐trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W‐cNTnC in presence of its biological binding partner troponin I peptide (cTnI_144–163) and in the absence of TFE. We have also characterized a panel of six F77W‐cNTnC structures in the presence and absence TFE, cTnI_144–163, and the extra mutation V82A, and used ¹⁹F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

pH-induced conformational change in an alpha-helical coiled-coil is controlled by His residues in the hydrophobic core

An alpha-helical coiled-coil structure is one of the basic structural units in proteins. Hydrophilic residues at the hydrophobic positions in the coiled-coil structure play important roles in structures and functions of natural proteins. We reported here a peptide that formed a triple stranded alpha-helical coiled-coil showing the pH-dependent structural change. The peptide was designed to have two His residues at the hydrophobic positions of the center of the coiled-coil structure. The peptide folded into a triple stranded coiled-coil at neutral pH, while it unfolded at acidic pH. This construct is useful to create a protein that the structure or function is controlled by pH.     

Solution structure of a small protein containing a fluorinated side chain in the core

We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe --> F(5)-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F(5)-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl-aryl or perfluoroaryl-perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 --> F(5)-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by approximately 1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe --> F(5)-Phe mutations offer the possibility of greater tertiary structural stability from side chain-side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability.     

Redesigning the hydrophobic core of a four-helix-bundle protein.

Rationally redesigned variants of the 4-helix-bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native-like physical properties of the wild-type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild-type Rop. The equilibrium stability of the repacked proteins to thermal denaturation is enhanced relative to that of the wild-type protein. The rate of chemically induced folding and unfolding of wild-type Rop is extremely slow when compared with other small proteins. Interestingly, although the repacked proteins are more thermally stable than the wild type, their rates of chemically induced folding and unfolding are greatly increased in comparison to wild type. Perhaps as a consequence of this, their equilibrium stabilities to chemical denaturants are slightly reduced in comparison to the wild type.     

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.     

Alkylglucosides with isoprenoid-type hydrophobic chains-effects of hydrophobic chain size on the aqueous phase behavior

Aqueous phase diagrams were constructed for two new alkylglucosides with isoprenoid-type hydrophobic chains, viz. 1-O-beta-(3,7-dimethyloctyl)-D-glucopyranoside, beta-Glc(Ger), and 1-O-beta-(3,7,11,15-tetramethylhexadecyl)-D-glucopyranoside, beta-Glc(Phyt). In a low concentration regime, from 0.17 to 34 wt.% beta-Glc(Ger), the beta-Glc(Ger)/water system exhibits two phase, a dilute (L1dil) and a concentrated isotropic phase (L1con), coexistence region. Above about 62 wt.% beta-Glc(Ger), an Lalpha phase is formed. The extent of the L1dil + L1conc two-phase region decreases as temperature increases and totally disappears above 130 degrees C, exhibiting an upper critical temperature. The beta-Glc(Phyt)/water system exhibits an Lalpha phase above 78 wt.% surfactant below which, an Lalpha + water two-phase region appears. One notable feature of these compounds is their low values of Krafft-eutectic temperature, TK, e.g. the value of TK for beta-Glc(Phyt) is below 0 degrees C although the total number of carbon atoms in the hydrophobic chain is as large as 20.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions

A complete series of configurationally isomers (L -L , L -D , D -L AND D -D ) of a dipeptide Leu-Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz ¹H NMR, the L -D and D -L isomers, but not hte L -L and D -D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu-βCH₂ and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe-βCH₂, the NOE enhancement observed between Leu-δCH₃ and Phe-phenyl groups revealed that these groups are in close proximity. These data indicated that L -D and D -L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac-Try-OEt as a substrate, the L -L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D -L isomer was strongest with K_i of 2.2 × 10_?5 M . It was found that the D -L isomer was only slowly hydrolysed but the L (or D )-D isomer was not. H-D -Phe-L -Leu-OBzl with the inverse sequence of H-D -Leu-L -Pre-OBzl inhibited chymotrypsin more strongly (K_i = 6.3 × 10^?6 M ). Since the free acid analogue of the D -L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester-benzyl group fits the S₁ site of chymotrypsin, while the side chain-side chain complexing hydrophobic core fits the S₂ site.     

Conformational modification of the PRP-hexapeptide by a direct covalent attachment of aromatic side chain groups

PRP-hexapeptide possessing the azo-bridge between Tyr1 and Phe5 residues, called azo-PRP-hexapeptide: (formula; see text), was synthesized and tested for immunoregulatory activity. High biological activity of the synthesized azo-PRP-hexapeptide suggests that the biologically active conformation of PRP-hexapeptide must be such that both aromatic rings (Tyr and Phe) are apparently close to each other.     

Side chain burial and hydrophobic core packing in protein folding transition states

A critical step in the folding pathway of globular proteins is the formation of a tightly packed hydrophobic core. Several mutational studies have addressed the question of whether tight packing interactions are present during the rate-limiting step of folding. In some of these investigations, substituted side chains have been assumed to form native-like interactions in the transition state when the folding rates of mutant proteins correlate with their native-state stabilities. Alternatively, it has been argued that side chains participate in nonspecific hydrophobic collapse when the folding rates of mutant proteins correlate with side-chain hydrophobicity. In a reanalysis of published data, we have found that folding rates often correlate similarly well, or poorly, with both native-state stability and side-chain hydrophobicity, and it is therefore not possible to select an appropriate transition state model based on these one-parameter correlations. We show that this ambiguity can be resolved using a two-parameter model in which side chain burial and the formation of all other native-like interactions can occur asynchronously. Notably, the model agrees well with experimental data, even for positions where the one-parameter correlations are poor. We find that many side chains experience a previously unrecognized type of transition state environment in which specific, native-like interactions are formed, but hydrophobic burial dominates. Implications of these results to the design and analysis of protein folding studies are discussed.     

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.     

The remarkable hydrophobic effect of a fatty acid side chain on the microbial growth promoting activity of a synthetic siderophore

The ability of synthetic derivatives of the siderophore tripeptide of N ⁵-hydroxy-N ⁵-acetyl-l-ornithine to promote the growth of various strains of mycobacteria and Gram negative bacteria was found to depend significantly on the hydrophobic nature of the derivative. Although the tripeptide of N ⁵-hydroxy-N ⁵-acetyl-l-ornithine is not normally utilized by mycobacteria, an N-terminal palmitoyl derivative mimicked natural mycobactin J in all studies.     

Structure of a designed, right-handed coiled-coil tetramer containing all biological amino acids

The previous design of an unprecedented family of two-, three-, and four-helical, right-handed coiled coils utilized nonbiological amino acids to efficiently pack spaces in the oligomer cores. Here we show that a stable, right-handed parallel tetrameric coiled coil, called RH4B, can be designed entirely using biological amino acids. The X-ray crystal structure of RH4B was determined to 1.1 Angstrom resolution using a designed metal binding site to coordinate a single Yb(2+) ion per 33-amino acid polypeptide chain. The resulting experimental phases were particularly accurate, and the experimental electron density map provided an especially clear, unbiased view of the molecule. The RH4B structure closely matched the design, with equivalent core rotamers and an overall root-mean-square deviation for the N-terminal repeat of the tetramer of 0.24 Angstrom. The clarity and resolution of the electron density map, however, revealed alternate rotamers and structural differences between the three sequence repeats in the molecule. These results suggest that the RH4B structure populates an unanticipated variety of structures.     

Site-specific modification of de novo designed coiled-coil polypeptides with inorganic redox complexes.

A stepwise procedure for preparing of site-specific binuclear metallopeptides is described. The modification procedure involves the alkylation of a cysteine side chain by reaction with [Ru(bpy)(2)(phen-ClA)](2+), where bpy = 2,2'-bipyridine and phen-ClA = 5-chloroacetamido-1,10-phenanthroline, followed by the coordination of a ruthenium pentammine complex to a histidine residue located elsewhere along the sequence. The apo and metalated versions of the peptides C10H21(30-mer) and H10C21(30-mer) display circular dichroism spectra having minima at 208 and 222 nm, with theta(222)/theta(208) = 1.04 to indicate that these peptides exist as alpha-helical coiled-coils in aqueous solution. When the ruthenium polypyridyl complex is attached to C10H21(30-mer), the Delta-l and Lambda-l diastereomers of the resulting metallopeptide can be readily separated from each other by reversed-phase HPLC. However, in the case of the related H10C21(30-mer) metallopeptide, the two diastereomers cannot be chromatographically resolved. These results indicate how the subtle interplay between peptide conformation/sequence and metal complex geometry may alter some of the physical characteristics of metallopeptides.