Intracellular environment can change protein conformational dynamics in cells through weak interactions

Abstract

Conformational dynamics is important for protein functions, many of which are performed in cells. How the intracellular environment may affect protein conformational dynamics is largely unknown. Here, loop conformational dynamics is studied for a model protein in Escherichia coli cells by using nuclear magnetic resonance (NMR) spectroscopy. The weak interactions between the protein and surrounding macromolecules in cells hinder the protein rotational diffusion, which extends the dynamic detection timescale up to microseconds by the NMR spin relaxation method. The loop picosecond to microsecond dynamics is confirmed by nanoparticle-assisted spin relaxation and residual dipolar coupling methods. The loop interactions with the intracellular environment are perturbed through point mutation of the loop sequence. For the sequence of the protein that interacts stronger with surrounding macromolecules, the loop becomes more rigid in cells. In contrast, the mutational effect on the loop dynamics in vitro is small. This study provides direct evidence that the intracellular environment can modify protein loop conformational dynamics through weak interactions.

Fig. 1.. Protein backbone amide relaxation rates.

¹⁵N R₁ (A), R₂ (B), and ¹⁵N-{¹H} NOE (C) were measured for GB3L at both 600 and 900 MHz fields in the buffer and in E. coli cells at 303 K. The pH in cells, monitored by using ¹⁵N chemical shift of H33 (fig. S1), was adjusted to be the same as that in the buffer (40 mM bis-tris propane/40 mM Hepes, pH 6.8). Error bars are SEM (n = 3 independent experiments).

Fig. 2.. Conformational dynamics of GB3L.

(A) Order parameters in the buffer only, with anionic SNPs (aSNP), and in E. coli cells. The presence of anionic SNPs extends the dynamic observation time window so that picosecond to microsecond dynamics (S²_SNP) can be detected, which is undetectable in the conventional model-free analysis of ¹⁵N R₁, R₂, and NOE data measured in the buffer only (S²_buf). GB3L S² in E. coli cells (S²_cell) agrees better with S²_SNP, suggesting that the dynamics detected in cells is probably on picosecond to microsecond timescale. Picosecond to millisecond timescale S² fitted from backbone amide ¹H-¹⁵N RDCs (S²_RDC) was also included (only residues with at least two sets of experimental RDCs are shown). (B) Correlation of S² in E. coli cells (S²_cell) measured at 600 and 900 MHz fields. Two residues D19 and T21 show conformational exchanges (in red). Error bars (except for S²_RDC) are SEM (n = 3 independent experiments). Error bars for S²_RDC are SD from 10 independent ensemble fittings.

Fig. 3.. Ensemble fitting of GB3L backbone ¹H-¹⁵N RDCs.

A total of four sets of RDCs were measured at 900 MHz, with three from DO3MA-6MePy-Tm³⁺ attached to K24C, V26C, or A53C, and one from the external alignment medium Pf1. The x-ray structure of GB3 [pdb code: 2OED (87)] with the inserted loop was used as the starting structure for molecular dynamic simulation to generate an ensemble of eight structures for the experimental RDC fitting. (A) Correlation between experimental and back-calculated RDCs from the ensemble, and singular value decomposition of experimental RDCs, which was used to yield singular values (panel insert) and check the independence of RDCs (see more details in the main text). (B) Ensemble averaged structure displayed with the pointed DO3MA-6MePy-Tm³⁺ attaching sites. The order parameter S²_RDC is displayed in Fig. 2A for comparison with S² from the other methods.

Fig. 4.. ¹⁵N CPMG NMR relaxation dispersion data analysis for GB3L.

The data were measured in the buffer of 40 mM bis-tris propane/40 mM Hepes, pH 6.8, at 303 K. A two-site exchange model was used to fit the CPMG data for 25 residues that show chemical exchanges. (A) CPMG profile of A39 and its fitting curves. (B) Correlation between Δω_CPMG, the chemical shift difference from the fitting, and Δω_F-U, the chemical shift difference (absolute value) between the folded and unfolded GB3L. (C) Reduced χ² error versus the fitted unfolded state population. (D) χ² error versus the fitted exchange rate. The positive correlation between Δω_CPMG and Δω_F-U (absolute value) indicates that the conformational exchange corresponds to the protein folding and unfolding process. Error bars are SEM (n = 3 independent experiments).

Fig. 5.. GB3L conformational dynamics and its sequence dependence.

The loop 1 sequence was varied by changing X in GXSGG to different amino acids. (A) S² order parameters for different mutants, F, L, V, K, S, and D. (B) Averages of loop S² (including G11, G14, G15, T16, and L17, which have S² values in all GB3L variants) of different mutants in the buffer only, in the presence of anionic SNP, and in cells. The loop S² average in cells (C) is correlated with <ΔR₂> [ΔR₂ = R₂(cell) − R₂(buffer)] averaged over rigid GB3L residues. Error bars are SEM (n = 3 independent experiments).

Fig. 6.. Impact of SNPs on the conformational dynamics.

(A) Order parameters S² of the GB3L K mutant measured in 40 mM bis-tris propane/40 mM Hepes, at pH 6.8 and 303 K. (B) The average S² of loop 1 is smaller with additional 200 mM lysine in the anionic SNPs (aSNP) at the same pH and temperature. A similar decrease was seen in the same buffer but with neutral SNPs (nSNP). It is apparent that the electrostatic attraction between the GKSGG loop and anionic SNPs suppresses the loop dynamics. Error bars are SEM (n = 3 independent experiments).

Fig. 7.. Order parameters derived from ensemble fitting of backbone ¹H-¹⁵N RDCs.

A total of four sets of RDCs were measured and fitted in the same way for GB3L. (A and C) Correlation between experimental and back-calculated RDCs (for K and V mutants) from the ensemble. (B and D) Dynamics (S²_RDC) calculated from the structure ensemble for the two mutants (only residues with at least two sets of RDCs are shown). S²_SNP is also displayed for comparison. Error bars are SD from 10 independent ensemble fittings.