Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase.
Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.
Severcan
Feride
F
Department of Biology, Middle East Technical University, 06531, Ankara, Turkey. feride@metu.edu.tr
Haris
Parvez I
PI
D000818:Animals; D002950:Citrate (si)-Synthase / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013552:Swine; D013696:Temperature
D002950:Citrate (si)-Synthase