Two-dimensional infrared correlation spectroscopy as a probe of sequential events in the thermal unfolding of cytochromes c.
The sequential unfolding events of horse, cow, and tuna ferricytochromes c (cyt c) as a function of increasing temperature over the range 25-81 degrees C were investigated by resolution-enhanced two-dimensional infrared (2D IR) correlation spectroscopy. The 2D IR analysis revealed that in the thermal denaturation of the two mammalian cyts, the overall sequence of unfolding is similar, with denaturation of extended-chain and turn structures occurring prior to unfolding of alpha-helices, followed by denaturation of residual stable extended-chain structures. In tuna cyt c, denaturation of all extended-chain structures precedes the unfolding of alpha-helices. Moreover, in cow cyt c, unfolding of all helical components occurs as one cooperative unit, but in horse and tuna cyts c, the helical components behave as subdomains that unfold separately, as proposed recently by Englander and co-workers for horse cyt c [Bai et al. (1995) Science 269, 192-197; Milne et al. (1999) J. Mol. Biol. 290, 811-822]. At higher temperatures, following the loss of secondary structure, protein aggregation occurs in the three cyts c. The data presented here establish that variations in the thermal unfolding of cyts c can be associated with specific sites in the protein that influence local flexibility yet have little affect on global stability. This study demonstrates the power of resolution-enhanced 2D IR correlation spectroscopy in probing unfolding events in homologous proteins.
Filosa
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Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.
Wang
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Ismail
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English
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D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D006358:Hot Temperature; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013055:Spectrophotometry, Infrared / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D014413:Tuna
D003574:Cytochrome c Group