Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy.

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.

Use of photoaffinity probes containing poly(ethylene glycol) spacers for topographical mapping of the cholecystokinin receptor complex.

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 A, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the Mr = 85,000-95,000 glycoprotein with Mr = 42,000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 A, membrane proteins of Mr = 80,000 and Mr = 40,000 were specifically labeled. The product of endo-beta-N-acetylglucosaminidase F digestion of the Mr = 80,000 protein was Mr = 65,000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the Mr = 85,000-95,000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of Mr = 80,000 and Mr = 40,000 may represent noncovalently associated subunits sited within 16.2 A of the binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)