FTIR study of the thermal denaturation of alpha-actinin in its lipid-free and dioleoylphosphatidylglycerol-bound states and the central and N-terminal domains of alpha-actinin in D2O.
Fourier transform infrared (FTIR) spectroscopy has been carried out to investigate the thermal denaturation of alpha-actinin and its complexes with dioleoylphosphatidylglycerol (DOPG) vesicles. The amide I regions in the deconvolved spectra of alpha-actinin in the lipid-free and DOPG-bound states are both consistent with predominantly alpha-helical secondary structure below the denaturation temperatures. Studies of the temperature dependence of the spectra revealed that for alpha-actinin alone the secondary structure was unaltered up to 40 degrees C. But, in the presence of DOPG vesicles, the thermal stability of the secondary structure of alpha-actinin increased to 55 degrees C. The thermal denaturation mechanisms of the lipid-free and DOPG-bound states of alpha-actinin also vary. The secondary structure of the lipid-free alpha-actinin changed to be predominantly unordered upon heating to 65 degrees C and above. Whereas, the original alpha-helical structure in the DOPG-bound alpha-actinin retained even at 70 degrees C, the highest temperature we examined. Analysis of the reduction in amide II intensities, which is due to peptide H-D exchange upon heating alpha-actinin in D2O, showed that partially unfolded states with increased solvent accessibility but substantial secondary structures could be observed from 35 to 40 degrees C only if DOPG vesicles were present. A so-called "protamine precipitation" method has been developed to purify the N-terminal domain of alpha-actinin by use of the fact that the central domain of alpha-actinin is negatively charged but the N-terminal domain is positively charged. Thermal denaturation of the central and N-terminal domains of alpha-actinin were then investigated with FTIR. The secondary structure of the N-terminal domain of alpha-actinin was found to be thermally sensitive below 35 degrees C, which is characterized as the increase of the alpha-helical structure at the expense of the random coil upon heating the N-terminal domain from 4 to 35 degrees C. The membrane-binding ability of the N-terminal domain of alpha-actinin was proposed in terms of the analysis of the local electrostatic properties of alpha-actinin and the assignment of the amide II bands in the FTIR spctra of alpha-actinin.
Department of Biophysics, Beijing Medical University, P. R. China. xh3f@virginia.edu
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D000185:Actinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D017666:Deuterium Oxide / Q000737:chemistry; D006358:Hot Temperature; D008081:Liposomes / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011479:Protamines / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012474:Salmon; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared
D008081:Liposomes; D008563:Membrane Lipids; D010446:Peptide Fragments; D010715:Phosphatidylglycerols; D010743:Phospholipids; D011479:Protamines; D012996:Solutions; D000185:Actinin; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D017666:Deuterium Oxide