The amphipathic alpha-helices of exchangeable apolipoproteins (apo) function to simultaneously facilitate interaction with lipid surfaces and the aqueous environment. In contrast to mammalian apoA-I's, which self-associate in the absence of lipid, chicken apoA-I, which shares 66% sequence homology with human apoA-I, exists as a monomeric protein when dissociated from high-density lipoprotein (HDL). Sedimentation equilibrium studies conducted in the analytical ultracentrifuge yielded a weight-average molecular weight of 28,170. Corresponding sedimentation velocity and diffusion experiments gave rise to s0(20,w) = 2.23 S and D0(20,w) = 6.39 x 10(-7) cm2/s. A translational frictional ratio (f/fmin) of 1.18 and an axial ratio of 4.0 were also determined from this data. The Stokes radius (Rs,sed = 2.80 nm) and translational frictional ratio were subsequently used to calculate estimated molecular dimensions of 25.2 x 100.8 A for chicken apoA-I. Circular dichroism (CD) studies revealed a highly alpha-helical structure predicted to be 74% by Provencher-Glöckner analysis. Denaturation studies performed on lipid-free apoA-I and monitored by CD revealed a midpoint of denaturation of 0.64 M guanidine hydrochloride. From plots of delta G(app) versus guanidine hydrochloride concentration, a delta GDH2O of 1.86 kcal/mol was determined. In other studies, a midpoint of temperature-induced denaturation for apoA-I of 57 degrees C was obtained. The effect of solvent pH on the secondary structure content of apoA-I revealed a significant loss of alpha-helix below pH 4.0 and above pH 10, suggesting that lipid-free apoA-I may by partially stabilized by the formation of intra- or interhelix salt bridges between oppositely charged amino acid side chains.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4558, 4559 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||3|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O temp:20.0 C ; Experimental Assay: m ; Experimental Assay: dG_H2O temp:25.0 C|
|Libraries||Mutations for sequence MRGVLVTLAVLFLTGTQARSFWQHDEPQTPLDRIRDMVDVYLETVKASGKDAIAQFESSAVGKQLDLKLADNLDTLSAAAAKLREDMAPYYKEVREMWLKDTEALRAELTKDLEEVKEKIRPFLDQFSAKWTEELEQYRQRLTPVAQELKELTKQKVELMQAKLTPVAEEARDRLRGHVEELRKNLAPYSDELRQKLSQKLEEIREKGIPQASEYQAKVMEQLSNLREKMTPLVQEFRERLTPYAENLKNRLISFLDELQKSVA|