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Conformational analysis and stability of collagen peptides by CD and by 1 H‐ and 13 C‐NMR spectroscopies
Author(s) -
Consonni Roberto,
Zetta Lucia,
Longhi Renato,
Toma Lucio,
Zanaboni Giuseppe,
Tenni Ruggero
Publication year - 2000
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(200001)53:1<99::aid-bip9>3.0.co;2-d
Subject(s) - chemistry , trimer , peptide , crystallography , carbon 13 nmr , proton nmr , aqueous solution , circular dichroism , monomer , stereochemistry , organic chemistry , dimer , polymer , biochemistry
Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1 H‐ and 13 C‐nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10°C in acidic aqueous solution by peptide αl(I) CB2 only, and to lesser extent, by α1(I) CB4, whereas peptides α1(I) CB5 and α2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides α1(I) CB2 and α1(I) CB4 only were able to form trimeric species at temperature between 14 and 20°C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that α1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1 H‐ and 13 C‐nmr spectra acquired in the temperature range 0–47 and 0–27°C, respectively, indicated that with decreasing temperature the most abundant form of α1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple‐helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1 H‐nmr following the change in area of the signal belonging to one of the two β protons of the C‐terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4°C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple‐helical backbone and side chains constitute a single unit. © 2000 John Wiley & Sons, Inc. Biopoly 53: 99–111, 2000