The Effects of Chain Length and Thermal Denaturation on Helix-Forming Peptides:  A Mode-Specific Analysis Using 2D FT-IR

Abstract

The major goal of this project was to use FT-IR spectroscopy to monitor the effects of chain length and temperature on small, helix-forming peptides of the general form, Ac-W(EAAAR) n A-NH 2 , where n ) 1, 3, 5, and 7, in aqueous solutions. FT-IR spectra were collected in D 2 O as a function of temperature in the range of -4 to 95°C. The spectral range of interest, 1500-1725 cm -1 , contains the amide-I′ band of the fully-exchanged H f D peptide bond. Even in these simple peptides, the amide-I′ region of the IR spectra is complex and congested, composed of features derived from the conformation of the peptide backbone and from the contributions of amino acid side chains. Unambiguous resolution of peak positions and intensities is thus extremely difficult, particularly when assessing subtle differences between two data sets. Two-dimensional correlation analysis (Noda, I. J. Am. Chem. Soc. 1989, 111, 8116. Noda, I. Applied Spectrosc. 1990 was used to guide and verify the results of the peak-fitting procedure and thereby facilitate physical interpretation of the temperature-dependent spectra. The results of the two-dimensional analysis and fitting procedure show that the spectral bands, particularly those of the amide-I′ band, exhibit significant frequency shifts and bandwidth and intensity changes as a function of temperature and chain length. For the amide-I′ modes arising from the helical and random conformations of the peptide bond, the normalization of peak intensities to units of molar absorptivity is discussed in terms of different models. Two different molar absorptivity calculations are presented, the first using the length-dependence of R-helical frequencies as predicted by perturbation theory, and the second assuming a more rigorous two-state transition. The results from each are discussed in terms of the effects of chain length on R-helix stabilization and in terms of a mechanism of helix unfolding.

Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

“…Although the synchronous spectrum is always required for interpretation, the asynchronous spectrum is of particular interest, because it permits the distinction of spectral intensity changes that occur out-of-phase (that is, delayed or accelerated), as a function of the applied perturbation (in our case, temperature). Similar 2D plots were used previously to illustrate the chain of events in the dissociation of amide hydrogen bonds in Nmethylacetamide (13), to improve the resolution of individual band components in the spectrum of myoglobin (14), to resolve the temperature-dependent spectra of helix-forming peptides (15), and to detect sudden changes in the hydration of ovalbumin that precede the unfolding of the protein (16). Very recently, Hochstrasser et al (17) reported a different form of 2D (nonlinear) IR spectroscopy in which perturbations to the system are introduced by ultrafast IR laser pulses, allowing the detection of ultrafast vibrational relaxation times in small model peptides, an approach more akin to 2D NMR.…”

Quantification of transition dipole strengths using 1D and 2D spectroscopy for the identification of molecular structures via exciton delocalization: Application to α-helices

“…For instance, random coils absorb at about 1645 cm −1 , which overlaps with α-helices that absorb between 1635 cm −1 and 1655 cm −1 , depending on whether they are soluble or membrane bound. [3][4][5][6][7][8] In principle, one can fit the amide I absorption band, but it is difficult to be confident in fits because structural disorder causes symmetry forbidden transitions to appear and because the absorption bands themselves have complex lineshapes, among other problematic issues. [9][10][11][12][13][14][15][16] In this paper, we report a method for measuring transition dipole strengths from 1D and 2D IR spectroscopy and illustrate the utility of using transition dipole strengths to identify secondary structure.…”

“…Non-linear spectroscopies are more sensitive to transition dipole strengths than are linear spectroscopies. The integrated area of absorption spectroscopies is independent of coupling because it scales as the transition dipole squared, e.g., |μ| 2 whereas 2D IR spectra scale as |μ| 4 . As a result, the integrated area of a 2D IR spectrum is not conserved when the coupling changes.…”

“…We take advantage of the fact that the amplitudes of both 1D and 2D spectra scale linearly with concentration, C, but non-linearly with transition dipole strengths. That is, 1D spectra scale as C|μ| 2 while 2D spectra scale as C|μ| 4 . This fact is well documented in the literature and has been used on occasion to approximate transition dipole strengths.…”

The application of two‐dimensional correlation spectroscopy to surface and interfacial analysis

“…160 -177 The denaturation and unfolding of proteins in solution 174,178 -183 and the thermal transitions of a number of proteins, such as cytochrome c, 172,184 CMP kinases, 181 ovalbumin, 174 -lactoglobulin, 185 avidin 186 and synthetic helix-forming peptides 187 have been investigated using generalized 2D IR correlation spectroscopy. Studies have also been published that use pH gradients or H/D exchange to enhance the amide spectral region and assign conformations to the underlying band components.…”