Protein Dynamics Influence the Enzymatic Activity of Phospholipase A/Acyltransferases 3 and 4.

Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.

Removal of slow-pulsing artifacts in in-phase 15N relaxation dispersion experiments using broadband 1H decoupling.

Understanding of the molecular mechanisms of protein function requires detailed insight into the conformational landscape accessible to the protein. Conformational changes can be crucial for biological processes, such as ligand binding, protein folding, and catalysis. NMR spectroscopy is exquisitely sensitive to such dynamic changes in protein conformations. In particular, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are a powerful tool to investigate protein dynamics on a millisecond time scale. CPMG experiments that probe the chemical shift modulation of 15N in-phase magnetization are particularly attractive, due to their high sensitivity. These experiments require high power 1H decoupling during the CPMG period to keep the 15N magnetization in-phase. Recently, an improved version of the in-phase 15N-CPMG experiment was introduced, offering greater ease of use by employing a single 1H decoupling power for all CPMG pulsing rates. In these experiments however, incomplete decoupling of off-resonance amide 1H spins introduces an artefactual dispersion of relaxation rates, the so-called slow-pulsing artifact. Here, we analyze the slow-pulsing artifact in detail and demonstrate that it can be suppressed through the use of composite pulse decoupling (CPD). We report the performances of various CPD schemes and show that CPD decoupling based on the 90x-240y-90x element results in high-quality dispersion curves free of artifacts, even for amides with high 1H offset.