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What's in your buffer? Solute altered millisecond motions detected by solution NMR.

Biochemistry (2013-09-03)
Madeline Wong, Gennady Khirich, J Patrick Loria
ABSTRACT

To date, little work has been conducted on the relationship between solute and buffer molecules and conformational exchange motion in enzymes. This study uses solution NMR to examine the effects of phosphate, sulfate, and acetate in comparison to MES- and HEPES-buffered references on the chemical shift perturbation and millisecond, chemical, or conformational exchange motions in the enzyme ribonuclease A (RNase A), triosephosphate isomerase (TIM) and HisF. The results indicate that addition of these solutes has a small effect on (1)H and (15)N chemical shifts for RNase A and TIM but a significant effect for HisF. For RNase A and TIM, Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, however, show significant solute-dependent changes in conformational exchange motions. Some residues show loss of millisecond motions relative to the reference sample upon addition of solute, whereas others experience an enhancement. Comparison of exchange parameters obtained from fits of dispersion data indicates changes in either or both equilibrium populations and chemical shifts between conformations. Furthermore, the exchange kinetics are altered in many cases. The results demonstrate that common solute molecules can alter observed enzyme millisecond motions and play a more active role than what is routinely believed.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
HEPES, ≥99.5% (titration)
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SAFC
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