Circular dichroism of designed peptide helices and β-hairpins: analysis of trp- and tyr-rich peptides

VCD versus ECD spectroscopy. Peptides rich in aromatic residues yield anomalous far-UV electronic circular dichroism (ECD) spectra that preclude secondary structure assignment. The utility of vibrational circular dichroism (VCD) in conformation analysis is demonstrated by using a set of well-defined peptide helices and hairpins containing proximal aromatic residues

Methionine mutations of outer membrane protein X influence structural stability and beta-barrel unfolding.

We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability

Non-Protein Amino Acids in the Design of Secondary Structure Scaffolds

The use of stereochemically constrained amino acids permits the design of short peptides as models for protein secondary structures. Amino acid residues that are restrained to a limited range of backbone torsion angles (ϕ-ψ) may be used as folding nuclei in the design of helices and β-hairpins. α-Amino-isobutyric acid (Aib) and related Cαα dialkylated residues are strong promoters of helix formation, as exemplified by a large body of experimentally determined structures of helical peptides. DPro-Xxx sequences strongly favor type II’ turn conformations, which serve to nucleate registered β-hairpin formation. Appropriately positioned DPro-Xxx segments may be used to nucleate the formation of multistranded antiparallel β-sheet structures. Mixed (α/β) secondary structures can be generated by linking rigid modules of helices and β-hairpins. The approach of using stereochemically constrained residues promotes folding by limiting the local structural space at specific residues. Several aspects of secondary structure design are outlined in this chapter, along with commonly used methods of spectroscopic characterization

Influence of protein-micelle ratios and cysteine residues on the kinetic stability and unfolding rates of human mitochondrial VDAC-2.

Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 - 13,000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol(-1) for full-length hVDAC-2 and ∼23 kcal mol(-1) for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis

NMR analysis of aromatic interactions in designed peptide β-hairpins

Designed octapeptide β-hairpins containing a central DPro-Gly segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel β-strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel β-sheets, in the non-hydrogen bonding position (Ac-L-Y-V-DP-G-L-Y/W-V-OMe: peptides 1 and 2), diagonally across the strands (Boc-Y/W-L-V-DP-G-W-L-V-OMe: peptides 3 and 6), and along the strands at positions i and i + 2 (Boc-L-L-V-DP-G-Y-L-W-OMe: peptide 4). Two peptides served as controls (Boc-L-L-V-DP-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-DP-G-L-L-V-OMe: peptide 7) for aromatic interactions. All studies have been carried out using solution NMR methods in CDCl3 + 10% DMSO-d6 and have been additionally examined in CD3OH for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces

Differential scanning calorimetric analysis.

<p>DSC data for OmpX^HN (A and C; black and green) and OmpX^M (B and D; red and brown), generated using heat shock folding (A and B) or slow refolding (C and D) methods. Unfolding T_m and ΔH derived from fits to a two-state model, are indicated beside each curve.</p

OmpX^HN and OmpX^M show comparable refolding efficiency.

<p>(A) Cartoon representation of OmpX^HN (PDB ID: 1QJ8) generated using PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079351#pone.0079351-Schrodinger1" target="_blank">[62]</a>, with the three Met residues at positions 18, 21 and 118, rendered as spheres. These methionines have been mutated to leucine in OmpX^M. (B) SDS-PAGE analysis of unboiled samples, comparing the refolding efficiency of OmpX^HN (labeled HN) and OmpX^M (labeled M). Refolding of these samples was achieved by the slow folding method at 40°C, in lipids and detergents mentioned above each lane. Upon folding, OmpX migrates at ∼22 kDa, compared to the unfolded protein (labeled urea) at ∼16 kDa. Fraction folded (f_F), determined by densitometry using band intensity of the monomeric species, is indicated below each lane. Samples were not centrifuged to remove any precipitated protein, so that a correct estimate of the folding efficiency in each condition could be obtained. Note the formation of higher order oligomers for both proteins, upon refolding, as observed earlier for OmpA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079351#pone.0079351-Wang1" target="_blank">[63]</a>. LDAO: lauryldimethylamine oxide; DPC: n-dodecylphosphocholine; 60PC: 6∶0 diether PC; DMPC: 1,2 dimyristoyl-sn-glycero-3-phosphocholine; L: protein MW marker; Mo: unfolded monomer; Mo*: folded monomer; Di: unfolded dimer; Di*: folded dimer. (C) Fluorescence emission (top panels) and far-UV CD spectra (bottom panels) of OmpX^HN (left) and OmpX^M (right), refolded using heat shock (RF-HS) and slow folding at 40°C (RF-40). Spectra of unfolded protein samples (UF) in 8 M GdmHCl or directly re-suspended in buffer, for fluorescence and CD scans, respectively, are also provided for comparison. The labels reflect color codes used for the respective samples. Note that in the top left panel, the fluorescence emission spectrum of RF-HS OmpX^HN (black curve) is directly underneath the spectrum of RF-40 OmpX^HN (red curve).</p