Native-like structure and self-association behavior of apolipoprotein A-I in a water/n-propanol solution.
The effect of n-propanol on the secondary and tertiary structure of human apolipoprotein A-I (apoA-I), an interfacial protein, was investigated using near and far ultraviolet (UV)-circular dichroism (CD) and fluorescence spectroscopy, as well as limited proteolytic digestion with trypsin, and cross-linking with bis(sulfosuccinimidyl) suberate. The structure of apoA-I in n-propanol (30%, v/v) was compared with that in Tris buffer and in reconstituted, spherical or discoidal, high density lipoproteins (rHDL). Addition of n-propanol to apoA-I in Tris buffer induces major changes in its near and far CD spectra: alpha-helical content increases by 27% and the near UV-CD spectrum becomes very similar to that of apoA-I in rHDL particles. Fluorescence spectral, lifetime, and polarization results, and quenching by KI confirm that major structural changes occur in the N-terminal half of apoA-I as n-propanol is added: the Trp residues become more exposed to solvent than in buffer alone or in rHDL. Higher concentrations of guanidine hydrochloride or urea are required to denature apoA-I in n-propanol than in buffer alone, but a similar free energy of unfolding is observed. The N-terminus of apoA-I is relatively resistant to trypsin digestion and the C-terminus has equivalent digestion sites for apoA-I in the three states, but the kinetics of digestion are much slower in n-propanol and in rHDL compared to apoA-I in Tris buffer. Cross-linking experiments reveal that dimers of apoA-I exist in n-propanol, in contrast to dimers plus multimeric aggregates in Tris buffer. From these results we conclude that in 30% n-propanol the structure of apoA-I approaches that of 'native' lipid-bound apoA-I, in contrast to its structure in the aqueous Tris buffer.
Leroy
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Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign 61801.
Jonas
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D000433:1-Propanol; D016632:Apolipoprotein A-I / Q000737:chemistry*; D002942:Circular Dichroism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013388:Succinimides / Q000737:chemistry; D014357:Trypsin; D014867:Water
D016632:Apolipoprotein A-I; D012996:Solutions; D013388:Succinimides; D014867:Water; D000433:1-Propanol; C035760:bis(sulfosuccinimidyl)suberate; D014357:Trypsin