Protein Dynamics Studied by NMR Spin Relaxation. Conformational Transitions of a Calmodulin Mutant

Sammanfattning: Binding of calcium to the protein calmodulin leads to molecular reorganization that enables interaction with target peptides and activation of downstream processes. I have studied the dynamics of the calcium-loaded form of a C-terminal calmodulin mutant (E140Q-Tr2C) using NMR spin relaxation experiments. E140Q-Tr2C exhibits global conformational exchange on the microsecond time-scale. The populations of the two major exchanging states are very similar and previous studies have demonstrated that they resemble the calcium-free and calcium-loaded forms of the wildtype protein. The rate of interconversion is similar to the calcium off-rate in calmodulin, which makes E140Q-Tr2C an interesting model system for characterization of the molecular switch in calmodulin. While previous relaxation studies have focused on backbone dynamics, using the 15N chemical shift as a probe of exchange, I have extended the characterization to 1HN and 13Ca. I have also studied the correlated dynamics of the 15N-1H amide groups and 13Ca-13Ca on adjacent residues.I studied the correlated chemical exchange of backbone amide groups using differential multiple-quantum relaxation. By performing the experiments at three different static magnetic field strengths I was able to separate the exchange contribution from the other contributions to differential multiple-quantum relaxation. I conclusively show that the chemical exchange is fast on the chemical shift time. Using previously determined values for tex, I calculated the products of the chemical shift differences for 15N and 1H. The correlation with the wildtype model was very reasonable. In a subsequent study, I measured 1H amide proton dynamics using rotating-frame spin relaxation experiments. 1H rotating-frame relaxation is extremely useful for characterization of microsecond dynamics since the range of spin-lock fields that can be obtained greatly exceeds the range achievable for 15N. The average exchange correlation times agreed well with those previously determined by 15N experiments, with average values of 19 ± 7 ms and 21 ± 3 ms, respectively. The data could be modeled reasonably well by global two-state exchange with a common exchange correlation time of 19 ± 1 ms. By assuming equal populations of the two states I calculated the chemical shift differences. I show that the order of correlation with the wildtype model decreases in the order 15N > 15N-1H > 1H, which indicates that the 1H chemical shifts are more sensitive than the 15N shifts to variations in structure.The last two projects focus on 13Ca dynamics. In contrast to 15N and 1H, the 13Ca chemical shift is dominated by the dihedral angles f and y and is thus sensitive to fluctuations of secondary structure. I developed a new 13Ca rotating-frame experiment for characterization of exchange correlation times and amplitudes. A surprisingly large number of residues also show chemical exchange for 13Ca and the average value of tex was 27 ms, in good agreement with previous data. The chemical shift differences suggests that the exchange is restricted to fluctuations with in a single region of the Ramachandran f-y space. In a complementary study I measured the correlated dynamics of 13Ci-1a-13Cia. Correlated exchange was verified only for a small subset of the residues. Interestingly, significant exchange was identified for virtually all pairs of the analyzed residues in helix F. I propose that this reflects a transient correlated fraying of this helix.