Two-dimensional infrared correlation spectroscopy as a probe of sequentialevents in the thermal unfolding of cytochromes c

The sequential unfolding events of horse, cow, and tuna ferricytochromes ( (cyt c) as a function of increasing temperature over the range 25-81 degree sC 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 occurri ng prior to unfolding of a-helices, followed by denaturation of residual st able extended-chain structures. In tuna cyt c, denaturation of all extended -chain structures precedes the unfolding of cr-helices. Moreover, in cow cy t c, unfolding of all helical components occurs as one cooperative unit, bu t in horse and tuna cyts c, the helical components behave as subdomains tha t unfold separately, as proposed recently by Englander and co-workers for h orse cyt ( [Bai ct al, (1995) Science 269, 192-197; Milne et al, (1999) J. Mel. Biol, 290, 811-822]. At higher temperatures, following the loss of sec ondary 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 demonstr ates the power of resolution-enhanced 2D IR correlation spectroscopy in pro bing unfolding events in homologous proteins.