Conformational studies of the N‐terminal lipid‐associating domain of human apolipoprotein C‐I by CD and ¹H NMR spectroscopy

Abstract

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 'H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-GI5 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (<2 h) of backbone amide protons with deuterium. The mobility of K12-GI5 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125 ' , centered at K12/E13, and 150", centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, 126, and 129, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.

Structural studies of a baboon (Papio sp.) plasma protein inhibitor of cholesteryl ester transferase

“…7!~;1608 centered near T17!, there is no evidence for slow conformational exchange in this region. One interpretation of these observations is that CETIP is bound to the surface of the micelle more tightly than apoC-I~1-38!, and consequently, CETIP also does not interact with the aqueous milieu as strongly as apoC- I~1-38!~Spyracopoulos & O'Neil, 1994;Rozek et al, 1997Rozek et al, , 1999 If apoC-I~1-38!, at least around K12-G15, does not bind to the micelle surface as tightly as CETIP, what are the possible physical reasons for the differences in lipid association and can the differences in lipid association account for the observed differences in CET inhibition? Human apoC-I~1-38!…”

“…Such an analysis illustrates an increase in the helical content of CETIP from 14 to 56% upon the addition of SDS. An increase in helical content has been observed with several peptides and proteins upon the addition of SDS~Wu & Yang, 1978;Rozek et al, 1995Rozek et al, , 1997Rozek et al, , 1999Buchko et al, 1996aBuchko et al, , 1996bWang et al, 1996aWang et al, , 1996b!. Because the calculated mean molar ellipticity values are sensitive to many elements~Hennessey & Johnson, 1982;Sarver & Krueger, 1991!, the CD data were also analyzed using two parameters, R1 and R2, which are independent of inaccuracies in determining peptide concentrations as well as those caused by slight wavelength shifts~Bruch et al, 1991!.…”

NMR of Membrane-Associated Peptides and Proteins

“…Structural studies of apolipoproteins in micelles by NMR were pioneered by him in the early 1990s. Solution structures of several peptides from apoA-I, apoA-II, apoC-I, apoC-II, and apoE in SDS or DPC micelles were determined by 2D NMR techniques [51,52,[111][112][113][115][116][117]. These peptides indeed adopt amphipathic helical structures, providing support for the amphipathic helix model proposed by Segrest [118].…”

Conformational and Disorder to Order Transitions in Proteins: Structure / Function Correlation in Apolipoproteins

“…In fact, it has been proposed that in the presence of lipids, the process of peptide folding corresponds to an enthalpy driven process supported by the energy employed for water displacement (Rozek et al, 1997). Localized changes in secondary structure of a number of proteins have been found to be physiologically relevant (Chakrabartty & Baldwin, 1995;Meador et al, 1992).…”