We speculate that the close proximity of cTnI to cTnC is favourable for increasing the frequency of collisions between cTnC and the switch region, counteracting the reduced probability of collisions due to the incomplete opening of the N-lobe of cTnC in the cardiac isoform. We have used PRE to follow the conformational changes associated with binding of Ca2+ to troponin within the binary cTnC-cTnI complex. Using the site-specific placement of nitroxide spin labels in cTn, we have mapped the quaternary interactions and described the change in average distance between key regions of the cTnI inhibitory subunit and the cTnC Ca2+ binding subunit. Our PRE-NMR studies clearly show that the switch peptide is tightly bound within the N-lobe of cTnC in the presence of Ca2+, whereas the inhibitory region exhibits conformational freedom, but remains in the vicinity of the central linker region of cTnC. In the absence of Ca2+, the switch peptide is completely released from the N-lobe of cTnC. Upon release, it moves,10 A˚ towards the C-lobe of cTnC. However, we see no evidence of an alternative binding site on cTnC for the switch peptide in the Ca2+ free state. Our PRE measurements also show that the interaction of the N-region of cTnI with the Nutlin-3 structural Clobe of cTnC is, unsurprisingly, Ca2+ independent. The geometrical positioning of key functional regions of the TnI subunit with respect to TnC and the subsequent movement of TnI accompanying activation, for both skeletal and cardiac isoforms, has been the subject of many biochemical studies over the past two decades. From these studies, structural models describing the cascade of conformational changes arising from binding of Ca2+ to the N-lobe of cTnC have been proposed with the molecular details for the positioning of most functional regions of cTnI reinforced by the crystal structure of the cardiac Tn core structure made available in 2003. However, definitive supportive data, required to provide a more complete detailed description of these structural models and associated Ca2+ induced changes, is lacking, since there is no crystal structure of the cardiac complex in the low Ca2+ state. Determination of such a structure is likely to remain a significant challenge due to its dynamic nature. The p21 Ras signaling pathway is activated by stimulation of the T cell receptor and plays a critical role in the acute activation of naive T cells. Activation of Ras, via GTP loading by guanine nucleotide exchange factors such as the diacylglycerol dependent RasGRP1 or the phosphotyrosinebinding Grb2/SOS complex, results in the rapid activation of several downstream signaling pathways, including the ERK, JNK, and p38 MAP kinase pathways as well as PI3K-induced effectors. Both the MAP kinase and PI3K signaling pathways contribute to transcription of acute activationinduced genes such as IL-2 that are critical to CD4+ T cell function. Studies in recent years have demonstrated that Ras signaling is far more complex than previously appreciated. The functional effect of Ras activation can be influenced by the GEF activating Ras, the location of Ras activation, the duration and strength of Ras signaling, and the developmental stage of the T cell.