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

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.

Figure 1

Chemical shift perturbations caused by phosphate (A,D), sulfate (B,E), and acetate (C,F). Composite ¹H,¹⁵N chemical shifts (Δ) are shown on a per-residue basis (A–C). Residues with Δ values > 0.05 ppm, given by the horizontal red line, are mapped onto the RNase A structure as red spheres (D–F). Δ was calculated as described in the Methods section by equation (3)

Figure 2

Transverse relaxation rate constants for RNase A. The R₂ values with τ_cp = 0.625 milliseconds are shown for the reference (red), phosphate (green), sulfate (blue), and acetate (brown) samples.

Figure 3

¹⁵N-CPMG dispersion curves. Data for MES reference (red), phosphate (green), sulfate (blue), and acetate (brown) are shown for select residues indicated at the top of each graph.

Figure 4. Millisecond motions in RNase A

Amino acid residues with R_ex ranging from zero (gray) to 40 s⁻¹ (red, see color bar) are shown on a ribbon diagram of RNase A for MES reference (A), phosphate (B), sulfate (C), and acetate (D).

Figure 5

Solute-dependent R_ex changes. Differences in R_ex between MES-reference and phosphate (A), sulfate (B), and acetate (C) containing RNase A samples as a function of amino acid sequence. In D–E the changes (positive, blue and negative, red) are shown as spheres on the respective backbone trace of RNase A. Data is only shown in A–F in which the difference in R_ex values is > than 20% of the MES-reference sample.

Figure 6

Comparison of conformational exchange parameters. A comparison of exchange rate constants (k_ex) from the MES reference is given in A–C for phosphate, sulfate, and acetate respectively. ϕ_ex comparisons are shown for the same samples in D–F. All data were obtained at 600 MHz from fits to equation (1). The diagonal lines in each have a slope of 1 to indicate a perfect correlation between the data points. In D–F, the axes are shown as a log₁₀ scale for viewing purposes only.

Figure 7

Buffer-altered chemical shifts in TIM. (A) shows the residue-specific composite chemical shift changes determined from equation (3). Values of Δ > 0.02 ppm are shown as red spheres on the TIM monomer in (B). The active site loop 6 is colored cyan.

Figure 8

Transverse relaxation rates for V167P/W168E TIM in MES (red) and phosphate (blue) buffers. Relaxation rates were determined using the two point method with τ_cp = 0.625 and 0.0 ms in a TROSY-relaxation compensated CPMG experiment at a total relaxation delay of 20 ms. Uncertainties were determined from duplicate experiments.

Figure 9

Conformational exchange contributions for triosephosphate isomerase. In (A) R_ex values > 3 s⁻¹ are shown for MES buffered TIM. In (C), these residues are indicated as red spheres on the monomer structure of the TIM dimer. Panels (B) and (D) show analogous results for phosphate buffered TIM. The active site loop 6 is colored cyan to orient the reader.

Figure 10

¹H-¹⁵N Chemical shift comparison for HisF. TROSY spectra for HisF in HEPES buffer (blue) and phosphate buffer (red). Δ values as calculated with equation (3) are shown for each assignable residue.

Figure 11

Crystallographic identification of ion binding sites. (A) shows an electrostatic surface rendering of RNase A viewing into the active site cleft. B–D show the same orientation as in A with phosphate (B), sulfate (C), and acetate (D) bound near the P1 site. The electrostatic surface was generated with the APBS and VMD. The structures used for B–D, respectively are 5RSA, 1RN3, and 4RSD.