Kimberly A. Bolin

NMR structure of a minimized human agouti related protein prepared by total chemical synthesis.

The structure of the chemically synthesized C‐terminal region of the human agouti related protein (AGRP) was determined by 2D 1H NMR. Referred to as inimized gouti elated rotein, MARP is a 46 residue polypeptide containing 10 Cys residues involved in five disulfide bonds that retains the biological activity of full length AGRP. AGRP is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRPs exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 (MC3r, MC4r), and MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C‐terminal regions are ∼40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C‐terminal regions. The NMR structure of MARP reported here can be characterized as three major loops, with four of the five disulfide bridges at the base of the structure. Though its fold is well defined, no canonical secondary structure is identified. While previously reported structural models of the C‐terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, we find that Cys spacing is not sufficient to correctly determine the 3D fold of the molecule.

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHNα coupling constants

SummaryAlanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CαH-NH couplings (3JHNα) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the CαH region of 1H 2D NMR spectra. Quantitative determination of all possible 3JHNα values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining 3JHNα from NOESY spectra. Because 3JHNα may be determined from 2D peaks outside of the CαH region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that 3JHNα can be obtained for every residue in the helical peptide Ac-(AAAAK)3A-NH2. The resulting 3JHNα profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.

Native-like secondary structure in a peptide from the α-domain of hen lysozyme

Background: To gain insight into the local and nonlocal interactions that contribute to the stability of hen lysozyme, we have synthesized two peptides that together comprise the entire α -domain of the protein. One peptide (peptide 1–40) corresponds to the sequence that forms two α -helices, a loop region, and a small β -sheet in the N-terminal region of the native protein. The other (peptide 84–129) makes up the C-terminal part of the α -domain and encompasses two α -helices and a 3 10 helix in the native protein. Results: As judged by CD and a range of NMR parameters, peptide 1–40 has little secondary structure in aqueous solution and only a small number of local hydrophobic interactions, largely in the loop region. Peptide 84–129, by contrast, contains significant helical structure and is partially hydrophobically collapsed. More specifically, the region corresponding to helix C in native lysozyme is disordered, whereas regions corresponding to the D and 3 10 helices in the native protein are helical in this peptide. The structure in peptide 84–129 is at least partly stabilized by interactions between residues in the two helical regions, as suggested by further NMR analysis of three short peptides corresponding to the individual helices in this region of the native protein. Conclusions: Stabilization of structure in the sequence 1–40 appears to be facilitated predominantly by long-range interactions between this region and the sequence 84–129. In native lysozyme, the existence of two disulphide bonds between the N- and C-terminal halves of the α -domain is likely to be a major factor in their stabilization. The data show, however, that native-like secondary structure can be generated in the C-terminal portion of the α -domain by nonspecific and nonnative interactions within a partially collapsed state.