Investigation of Slow Dynamics in Proteins
Subtitle: NMR Pulse Sequence Development and Application in Triosephosphate Isomerase
By: Wenbo Li
Publisher: [publisher not identified]
Published: 2012
Place of publication: [New York, N.Y.?]
Language: eng
Description: The dynamics of proteins on the millisecond time scale are on the same time scale as typical catalytic turnover rates, and can sometimes be closely related to enzymes' functions. Solid state NMR, equipped with magic angle spinning, is a very good technique to detect such millisecond dynamics, because it is suitable for many protein systems such as membrane proteins, and the anisotropic interactions recoupled in the solid state NMR can supply valuable geometric information regarding the dynamics. In this thesis, I mainly focus on the developing new dynamics detection pulse sequences based on previous Centerband-Only Detection of Exchange (CODEX) experiment and applying CODEX experiments to an enzyme system, triosephosphate isomerase (TIM), for studying the function of the millisecond dynamics in catalysis. Two newly developed pulse sequences, Dipolar CODEX and R-CODEX use the 13C-15N (Dipolar CODEX) and 1H-13C or 1H-15N (R-CODEX) dipolar couplings to detect dynamics. Compared with the chemical shift anisotropy used in the CODEX experiment, the dipolar coupling has a more direct relationship with the molecular geometry and could be better for extracting geometric information regarding reorientations.

A special characteristic of the R-CODEX sequence is that the use of an R-type dipolar recoupling sequence can suppress the effect of 1H-1H homonuclear couplings. This approach paves the way to detect both the correlation time and reorientational angle of the dynamics in fully protonated samples. These two pulse sequences are tested by detecting the π flip motion of urea and methylsulfone imidazole. The R-CODEX experiment is compared with two other millisecond dynamics detection methods: 2D-exchange experiments and line-shape analysis, using the example of in crystalline L-phenylalanine hydrochloride. The millisecond ring flip motion of the aromatic ring in L-phenylalanine hydrochloride is characterized in detail for the first time. The comparison between these three methods shows that the R-CODEX experiment does not require a chemical shift change in the process of the motion and that it can detect the dynamics even if there is the peak overlap in the spectra. Triosephosphate isomerase (TIM) is a well-known highly efficient enzyme.

Its loop motion (loop 6) has been extensively studied and been proven to be correlated with product release and be a rate-limiting step for the catalysis. Another highly conserved loop near the active site, loop 7 also has large changes in dihedral angles during ligand binding. Its motion is suspected to be correlated with loop 6 based on mutant experiments and solution NMR studies. However, the core sequence of loop 7, YGGS, is missing in the solution NMR spectrum. We assigned the GG pair in loop 7 (G209-G210) using 1-13C, 15N glycine labeling and solid state NMR experiments, and detected the loop 7's motion using 1-13C glycine labeling and CODEX experiments. We found that loop 7's motional rate (300+/-100 s-1) at -10oC agrees well with previously detected motional rates of loop 6 extrapolated from higher temperatures using an Arrhenius plot. This suggeststhat the motion of loop 6 probably correlates with loop 7. At the same time, the line-shape analysis for another GG pair (G232-G233), which forms hydrogen bonds with the ligand, indicates a ligand release rate (400+/-100 s-1) similar to loop 7's rate, supporting the hypothesis that the ligand release is also probably correlated with the motion of loop 7 and loop 6.
Notes: Department: Chemical Physics.

Thesis advisor: Ann E. McDermott.

Thesis (Ph.D.)--Columbia University, 2012.