Distance Measurements Using RET

Flpfre13.7. Siiucain of mefitUa Id due oTielical mte. The dooor U ttypiopliui-19. uni tbc aoceptor is an N-tenninal dansyl group. Itedsad from Set 24. 

It is important to notice the assumptions used in calculating the distance. We assumed that the orientation fechx  

For a high degree of RET donor quenching (FiWPb 1), a small percentage of unlabeled acceptor can result in a large change in the calculated transfer efficiency (Problem 13.9). 

In distance measurements using RET, there is often concern about the effects of the orientation factor K. At present, there is no way to measure K, short of detomina-tion of the X-ray crystal structure, in which case the distance would be known and thus there would be no reason to use energy transfer. However, it is possible to set limits on K, which in turn sets limits on the range of possible D-A distances. These limits are determined from the anisotropies of the donor and acceptor, which reflect the extent of orientational avenguig toward the dynamic average of 

The value of df represents the depolarization factor due to segmental motion of the donor (di ) or acc or (di ), but not the dqrolaiization due to overall rotational diffusion of the protein. Overall rotational diffusion is not important because it does not change the D-A orientation. The values of n and n are often taken as the steady-state and fundamental anisotropies, respectively, of the donor or acceptor. If the donor and acceptor do n ot rotate relative to each other during the excited-state lifetime, then di =di = 1.0, and = 0 and 1 1= 4. If both D and A ate independently and rapidly rotating over all space, = Km =.  

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