PEDS Advance Access originally published online on July 12, 2009
Protein Engineering Design and Selection 2009 22(8):497-513; doi:10.1093/protein/gzp029
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This article appears in the following Protein Engineering issue: Amyloids Special Issue [View the issue table of contents]
Solution state structures of human pancreatic amylin and pramlintide
1Department of Chemistry, University of Washington, Seattle, WA 98195, USA 2Amylin Pharmaceuticals, 9373 Towne Centre Dr., San Diego, CA 92121, USA
3 To whom correspondence should be addressed. E-mail: andersen{at}chem.washington.edu
We have employed pramlintide (prAM) as a surrogate for hAM in CD and NMR studies of the conformational preferences of the N-terminal portion of the structure in media which do not provide long-lived monomeric solutions of hAM due to its rapid conversion to preamyloid β aggregate states. Direct comparison of hAM and prAM could be made under helix-formation-favoring conditions. On the basis of CD and NMR studies: (i) the Cys2–Cys7 loop conformation has a short-span of helix (Ala5–Cys7); (ii) the extent to which this helix propagates further into the sequence is medium-dependent; a helix from Ala5 through Ser20 (with end fraying from His18 onward) is observed in aqueous fluoroalcohol media; (iii) in 12+ vol.% HFIP, the amyloidogenic region of hAM forms a second helical domain (Phe23–Ser29); (iv) the two helical regions of hAM do not have any specific geometric relationship as they are connected by a flexible loop that takes different conformations and (v) although the extreme C-terminus is essential for bioactivity, it is found to be extensively randomized with conformer interconversions occurring at a much faster rate than that is observed in the remainder of the peptide sequence. Two NMR-derived structures of the 1–22 sequence fragment of hAM have been derived. The work also serves to illustrate improved methods for the NMR characterization of helices. A detailed quantitative analysis of the NOE intensities observed in aqueous HFIP revealed alternative conformations in the C-terminal portion of the common amylin helix, a region that is known to be involved in the biorecognition phenomena leading to amyloidogenesis. Even though the SNN sequence appears to be a flexible loop, the chemical shifts (and changes induced upon helix structuring) suggest some interactions between the loop and the amyloidogenic segment of hAM that occur on partial helix formation.
Keywords: amyloidogenesis/CD spectroscopy/chemical shift deviations/helix formation/NMR
Received June 1, 2009; revised June 1, 2009; accepted June 3, 2009.
Abbreviations: ADCs, anti-distance constraints are designated as low-bounds-only (LBO) constraints herein; CD, circular dichroism or dichroic; CSD, chemical shift deviation; CGRP, calcitonin gene-related peptide; DCs, distance constraints, these are indicated without the hydrogen designation as
iNi+3, which corresponds to the distance or NOE intensity between H of residue i and HN of residue i + 3; DMSO, dimethyl sulfoxide; GdmCl, guanidinium chloride; hAM, human pancreatic amylin (likewise, rAM is rat amylin); HFIP, hexafluoroisopropanol; NOE, Nuclear Overhauser Effect; R1 = [
]max or []191/[]min, a ratio of CD ellipticities in the 225–190 nm span; R2 = []221/[]min, a ratio of CD ellipticities in the 225–195 nm span; SA, simulated annealing; SDS, sodium dodecyl sulfonate; TFA, trifluoroacetic acid; TFE, β,β,β-trifluoroethanol; the usual one and three letter abbreviation for the natural amino acids are used without definition as are the acronyms for NMR experiments: COSY, HSQC, HMQC, NOESY, RELAY, ROESY and TOCSY.