No abstract

Exendin-4, a 39 amino acid peptide originally isolated from the oral secretions of the lizard Heloderma suspectum, has been shown to share certain activities with glucagon-like-peptide-1 (GLP-1), a 30 amino acid peptide. We have determined the structuring preferences of exendin-4 and GLP-1 by NMR in both the solution and dodecylphosphocholine (DPC) micelle-associated states. Based on both chemical shift deviations and the pattern of intermediate range NOEs, both peptides display significant helicity from residue 7 to residue 28 with greater fraying at the N-terminus. Thornton and Gorenstein [(1994) Biochemistry 33, 3532-3539] reported that the presence of a flexible, helix-destabilizing, glycine at residue 16 in GLP-1 was an important feature for membrane and receptor binding. Exendin-4 has a helix-favoring glutamate as residue 16. In the micelle-associated state, NMR data indicate that GLP-1 is less helical than exendin-4 due to the presence of Gly16; chemical shift deviations along the peptide sequence suggest that Gly16 serves as an N-cap for a second, more persistent, helix. In 30 vol-% trifluoroethanol (TFE), a single continuous helix is evident in a significant fraction of the GLP-1 conformers present. Exendin-4 has a more regular and less fluxional helix in both media and displays stable tertiary structure in the solution state. In the micelle-bound state of exendin-4, a single helix (residues 11-27) is observed with residues 31-39 completely disordered and undergoing rapid segmental motion. In aqueous fluoroalcohol or aqueous glycol, the Leu21-Pro38 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure and produces ring current shifts as large as 3 ppm. This tertiary structure is partially populated in water and fully populated in aqueous TFE. The Leu21-Pro38 segment of exendin-4 may be the smallest protein-like folding unit observed to date. When the Trp-cage forms, fraying of the exendin-4 helix occurs exclusively from the N-terminus; backbone NHs for the C-terminal residues of the helix display H/D exchange protection factors as large as 10(5) at 9 degrees C. In contrast, no tertiary structure is evident when exendin-4 binds to DPC micelles. An energetically favorable insertion of the tryptophan ring into the DPC micelle is suggested as the basis for this change. With the exception of exendin-4 in media containing fluoro alcohol cosolvents, NMR structure ensembles generated from the NOE data do not fully reflect the conformational averaging present in these systems. Secondary structure definition from chemical shift deviations may be the most appropriate treatment for peptides that lack tertiary structure.

No abstract

We report here the changes in the CD spectrum produced by inserting aminoisobutyric acid (Aib) residues into the helical domain of human pancreatic amylin. In order to examine this effect at comparable net fractional helicities, CD spectra were measured for each species during the course of a helicity titration by trifluoroethanol addition. The addition of live Aib residues gave results of particular interest. At low net fractional helicity, this Aib-rich system displays a diminished R + RIG* (circa 208 nm) rotational strength versus the less Aib-rich species. However, NMR data and comparisons of CD difference spectra suggest that fluoroalcohol-induced extension of the short Aib-rich helix is in the form of an a helix. Given the diminished intensity of the minimum at 208 nm at low net helicity when 310 conformations should contribute, we urge extreme caution in using a [t&22 :[&ss ratio smaller than unity as a diagnostic for 310 helices.

Interleukin 4 (IL-4) is secreted by activated T cells and pleiotropically modulates both B-and T-lymphocyte function. In murine helper (CD4+) T-cell clones IL-4 production appears to be regulated independently of interferon Y and interleukin 2. To determine whether production of these lymphokines is also differentially regulated in uncloned human T cells, we studied lymphokine production *iy normal human peripheral T cells and T-cell subsets after ii vitro polyclonal activation. After maximal induction of lymphokine expression, IL-4 mRNA was detectable in <5% of CD41 and 1-2% of unfractionated T cells, whereas '33% and 60% of CD4' cells expressed detectable mRNA for interferon y and interleukin 2, respectively. This finding correlated with dramatically lower production of IL-4 mRNA and protein than of interferon rand interleukin 2 by peripheral blood and tonsillar T cells. The helper-inducer (CD4' CD45R-) T-cell subset, which significantly enhances in vitro immunoglobulin production, accounted for the preponderance of IL-4 mRNA accumulation and protein production by CD41 T cells; nevertheless, cells with detectable IL-4 mRNA constituted <10% of the CD4' CD45R-subset. Limitation of IL-4 production to a comparatively small population of normal human T cells could selectively regulate the effects of this lymphokine in T-cell-mediated immune responses; such selective regulation may be a fundamental mechanism for restricting the potentially pleiotropic effects of certain tymphokines to appropriate responder cells.Interleukin 4 (IL-4) is a T-cell lymphokine that modulates lymphocyte function by promoting B-cell growth, regulating immunoglobulin isotype expression, and promoting T-cell growth and cytotoxicity (1)(2)(3)(4). Interferon y (IFN-'y) (5,6) and, to a lesser extent, interleukin 2 (IL-2) (6, 7), which are primarily produced by activated T cells, also have pleiotropic effects on lymphoid cells. In certain cases all three lymphokines may have similar or synergistic activities, such as the enhancement of T-cell-mediated cytotoxicity (4,8), whereas in other situations, such as the regulation of immunoglobulin isotype expression by IL-4 and IFN-y, differential or antagonistic effects have been demonstrated (2, 9, 10).These in vitro findings raise the question as to how lymphokine production by activated T cells is regulated in vivo to result in an integrated effective immune response. One mechanism to facilitate distinct T-cell effector functions would be to limit the production of a lymphokine to a particular subtype ofT cells. Such segregation ofCD4' T-cell lymphokine production has been proposed by Mosmann and co-workers (11, 12) from the observation that most murine CD4' clones produce either IL-4 or IFN-y and IL-2 in a mutually exclusive fashion. However, most human CD4' T-cell clones do not appear to conform to this pattern (9, 13), and there is evidence that the patterns of lymphokine production obtained in clones may be influenced by the conditions used during the cloning process (14). In addition, c...