Matthew A. Kubasik

Acceleration of Short Helical Peptide Conformational Dynamics by Trifluoroethanol in an Organic Solvent

It has been known since the 1960s that small amounts of the cosolvent 2,2,2-trifluoroethanol (TFE) can have dramatic effects on the conformational stability of peptides and proteins in solution. Recent reviews conclude that no single mechanism accounts for all of the observed effects of TFE on biomolecular structures. 3] As a minor cosolvent, TFE is known to enhance the helical content of short peptides predisposed towards helical conformations. At high concentrations, however, TFE appears to disrupt native protein structures. The primary mechanism for the helix-stabilizing effect on peptides is thought to be the displacement of water molecules, 5] which enhances intramolecular hydrogen bonding. (TFE is a larger molecule than water and is also known to be a better hydrogen-bond donor but a weaker hydrogen-bond accepter than water.) Enhanced water/TFE solvent structure is a second suggested mechanism for TFE’s enhancement of helical structure in short peptides. In this mechanism, the unfavorable energetic cost of disrupting solvent structure to solvate the exposed peptide backbone leads to greater helical conformational stability. A third mechanism is grounded in the suggestion that helix-stabilizing intramolecular electrostatic interactions are enhanced by the reduction in the solvent dielectric constant afforded by TFE, which elevates the importance of helix-stabilizing intramolecular electrostatics. However, the electrostatic significance of TFE as a cosolvent has been cast into doubt due to its lack of significant effects on stability versus pH curves for a 19-mer peptide. We have used a kinetics experiment to investigate the effect of TFE on the structural stability of a highly helical peptide system in the low-dielectric organic solvent methylene chloride (e~9). Our peptide is an a-aminoisobutyric acid (Aib) octamer, Fmoc-Aib8-OtBu (Scheme 1; Fmoc = fluorenylmethoxycarbonyl). Aib Oligomers are known to exist in stable 310 helices at the octamer and higher polymeric levels, ok. 10] Peptides containing Aib residues have shown remarkable thermal stability ; their helical conformations display resistance to “melting” at elevated temperatures. 310 helices make up about 10 % of the helices observed in protein crystal structures and are characterized by a i/i+3 hydrogen bonding pattern in contrast to the i/i+4 hydrogen bonding pattern of the more common ahelix. Scheme 1 shows the peptide structure studied in this work and its 310 hydrogen-bonding pattern.

FT-IR spectroscopy and density functional theory calculations of 13C isotopologues of the helical peptide Z-Aib6-OtBu.

Isotope-edited FT-IR spectroscopy is a combined synthetic and spectroscopic method used to characterize local (e.g., residue-level) vibrational environments of biomolecules. We have prepared the 3(10) helical peptide Z-Aib6-OtBu and seven (13)C-enriched analogues that vary only in the number and position(s) of (13)C═O isotopic enrichment. FT-IR spectra of these eight peptides solvated in the nonpolar aprotic solvent dichloromethane have been collected and compared to frequency, intensity, and normal mode results of DFT calculations. Single (13)C enrichment of amide functional groups tends to localize amide I vibrational eigenmodes, providing residue-specific information regarding the local environment (e.g., hydrogen bonding or solvent exposure) of the peptide bond. Double (13)C enrichment of Z-Aib6-OtBu allows for examination of interamide coupling between two labeled amide functional groups, providing experimental evidence of interamide coupling in the context of 3(10) helical structure. Although the calculated and observed interamide couplings of Z-Aib6-OtBu are a few cm(-1) and less, the eight peptides exhibit distinct infrared spectra, revealing details of interamide coupling and residue level vibrational environments.

19F NMR chemical shifts induced by a helical peptide

19F NMR spectra of two neutral, organic‐soluble helical peptide octamers, each labeled at its N terminus with either 4‐fluorobenzamide or 4‐trifluoromethylbenzamide, in solvents with widely varying dielectric constants have been observed. The peptides are oligomers of α‐aminoisobutyric acid (Aib), which is a residue known to form stable 310 helices in organic solution. In relation to the 19F NMR spectra of a control molecule, the peptide terminating in 4‐fluorobenzamide shows a solvent‐dependent downfield chemical shift of between ∼1.5 and ∼4 ppm, whilst the peptide terminating in 4‐trifluoromethylbenzamide shows only an ∼0.2 ppm chemical shift dependence on the solvent dielectric constant. The experimental observations were compared to calculated values of the electric field generated by the correlation of dipolar amide units through the peptide’s helical conformation. We find the chemical‐shift response of the 4‐fluorobenzamide group to the peptide’s calculated electric field is consistent with the magnitude of 19F chemical shift dispersion observed in proteins.

Conformation-specific spectroscopy of capped, gas-phase Aib oligomers: tests of the Aib residue as a 310-helix former.

The conformational preferences of a series of capped peptides containing the helicogenic amino acid aminoisobutyric acid (Aib) (Z-Aib-OH, Z-(Aib)2-OMe, and Z-(Aib)4-OMe) are studied in the gas phase under expansion-cooled conditions. Aib oligomers are known to form 310-helical secondary structures in solution and in the solid phase. However, in the gas phase, accumulation of a macrodipole as the helix grows could inhibit helix stabilization. Implementing single-conformation IR spectroscopy in the NH stretch region, Z-Aib-OH and Z-(Aib)2-OMe are both observed to have minor conformations that exhibit dihedral angles consistent with the 310-helical portion of the Ramachandran map (ϕ, ψ = -57°, -30°), even though they lack sufficient backbone length to form 10-membered rings which are a hallmark of the developed 310-helix. For Z-(Aib)4-OMe three conformers are observed in the gas phase. Single-conformation infrared spectroscopy in both the NH stretch (Amide A) and C[double bond, length as m-dash]O stretch (Amide I) regions identifies the main conformer as an incipient 310-helix, having two free NH groups and two C10 H-bonded NH groups, labeled an F-F-10-10 structure, with a calculated dipole moment of 13.7 D. A second minor conformer has an infrared spectrum characteristic of an F-F-10-7 structure in which the third and fourth Aib residues have ϕ, ψ = 75°, -74° and -52°, 143°, Ramachandran angles which fall outside of the typical range for 310-helices, and a dipole moment that shrinks to 5.4 D. These results show Aib to be a 310-helix former in the gas phase at the earliest stages of oligomer growth.

FT-IR Spectroscopy and Density Functional Theory Calculations of Carbon-13 Isotopologues of the Helical Peptide Z-AIB(6)-OTBU

Isotope-edited FT-IR spectroscopy is a combined synthetic and spectroscopic method used to characterize local (e.g., residue-level) vibrational environments of biomolecules. We have prepared the 310 helical peptide Z-Aib6-OtBu and seven 13C-enriched analogues which vary only in the number and position(s) of 13C=O isotopic enrichment. FT-IR spectra of these eight peptides solvated in the nonpolar aprotic solvent dichloromethane have been collected and compared to frequency, intensity, and normal mode results of DFT calculations. Single 13C enrichment of amide functional groups tends to localize Amide I vibrational eigenmodes, providing residue-specific information regarding the local environment (e.g., hydrogen bonding or solvent exposure) of the peptide bond. Double 13C enrichment of Z-Aib6-OtBu allows for examination of inter-amide coupling between two labeled amide functional groups, providing experimental evidence of inter-amide coupling in the context of 310 helical structure. Although the calculated and observed inter-amide couplings of Z-Aib6-OtBu are a few cm−1 and less, the eight peptides exhibit distinct infrared spectra, revealing details of inter-amide coupling and residue level vibrational environments.

Isotope-edited FT-IR Spectra of Hexamers of α−Aminoisobutyric Acid

Isotope-edited FT-infrared spectra of the Amide I region of hexamers of alpha-aminoisobutyric acid (Z-Aib6-OtBu) have been collected in order to explore the effects of 13C=O enrichment on the FT-IR spectra in the conformational context of 310 helices. Oligomers of Aib are known to adopt predominantly 310 helical structures, even at short peptide lengths. The Amide I band is sensitive to the details of peptide secondary structure, but the competency of this band to distinguish between alpha- and 310 helical secondary structure remains an open question. The 310 helix is shorter than an alpha-helix of the same number of residues and exhibits an i to i+3 hydrogen bonding pattern, instead of an i to i +4 pattern of the alpha-helix. These differences bring amide oscillators of a 310 helix slightly closer in space and in shorter periodicity of hydrogen-bonding partnership as compared to an alpha-helix. We have collected infrared spectra of isotopomers of hexamers of Aib (e.g., Z-Aib-Aib-Aib-Aib-Aib-Aib-OtBu and Z-Aib∗-Aib-Aib∗-Aib-Aib-Aib-OtBu, Aib∗ = 13C enrichment at ∗C=O) in dichloromethane (a nonpolar aprotic solvent) and methanol (a polar, protic solvent) to examine the effects of carbon-13 enrichment on the spectra. Differences between the spectral lineshapes and isotopically-induced shifts (∼40 cm−1) of the Amide I bands of these peptide isotopomers in these two solvents will be discussed.

Hydrogen/Deuterium Exchange Studies on Helical Peptides in a DMSO/MeOD Solvent System

Introduction Oligomers of α-aminoisobutyric acid are known to form 310 helices in solution and in the solid state. NMR and IR studies have attempted to establish the minimum length at which reliable 310 helical structures occur. Estimates for the critical length for helix formation have ranged from trimer (with a single intramolecular hydrogen bond) to octamer level. We have used a hydrogen/deuterium exchange experiment to compare the protection from exchange with solvent deuterons afforded by the 310 helical structure at the tetramer and octamer level. We are using DMSO as solvent and MeOD as a deuteron donor. Oligomers of Aib are known to be helical in DMSO and the literature contains MD studies of Aib oligomers in DMSO for comparison to our experiments. The structures of the two peptides used in this work are shown below. Dotted lines indicate i/i+3 hydrogen bonding of a canonical 310 helical conformation. Within a 310 helical conformation, each structure shows two “solvent-exposed” amide protons. The remaining amide protons might be expected to be protected from exchange through intra-molecular hydrogen bonding.