The cold denatured state of the C-terminal domain of protein L9 is compact and contains both native and non-native structure

Abstract

Cold denaturation is a general property of globular proteins, and the process provides insight into the origins of the cooperativity of protein folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the fact that the cold denatured state is normally difficult to access experimentally. Special conditions such as addition of high concentrations of denaturant, encapsulation into reverse micelles, the formation of emulsified solutions, high pressure, or extremes of pH have been applied, but these can perturb the unfolded state of proteins. The cold denatured state of the C-terminal domain of the ribosomal protein L9 can be populated under native-like conditions by taking advantage of a destabilizing point mutation which leads to cold denaturation at temperatures above 0 degrees C. This state is in slow exchange with the native state on the NMR time scale. Virtually complete backbone (15)N, (13)C, and (1)H as well as side-chain (13)C(beta) and (1)H(beta) chemical shift assignments were obtained for the cold denatured state at pH 5.7, 12 degrees C. Chemical shift analysis, backbone N-H residual dipolar couplings, amide proton NOEs, and R(2) relaxation rates all indicate that the cold denatured state of CTL9 (the C-terminal domain of the ribosomal protein L9) not only contains significant native-like secondary structure but also non-native structure. The regions corresponding to the two native alpha-helices show a strong tendency to populate helical Phi and Psi angles. The segment which connects alpha-helix 2 and beta-strand 2 (residues 107-124) in the native state exhibits a significant preference to form non-native helical structure in the cold denatured state. The structure observed in the cold denatured state of the I98A mutant is similar to that observed in the pH 3.8 unfolded state of wild type CTL9 at 25 degrees C, suggesting that it is a robust feature of the denatured state ensemble of this protein. The implications for protein folding and for studies of cold denatured states are discussed.

Figure 1

The I98A mutant undergoes cold denaturation. (A) Ribbon diagram of CTL9 (residues 58–149 of protein L9), Protein data bank entry 1DIV. The hydrophobic core residue I98 as well as the N and C-termini are labeled. The ribbon diagram was made using PyMol. (B) Thermal denaturation curve of I98A CTL9 monitored by CD at 222 nm in H₂O at pH 5.7. (C) 1D ¹H-NMR spectra of I98A CTL9 at different temperatures in 100% D₂O at pD 6.0 (uncorrected pH meter reading). Only the aromatic region is shown, resonance assignments are labeled.

Figure 2

¹⁵N-HSQC spectrum of the cold denatured state of I98A CTL9 with the assignments indicated. The spectrum was recorded at pH 5.7, 12 °C.

Figure 3

Plots of the difference in the ¹³C_α secondary shifts and the ¹³C_β secondary shifts for (A) The native state of wild-type CTL9, (B) the cold denatured state of I98A CTL9. Note that the scale is different in panel A and panel B. Assignments were obtained at pH 5.7, 12 °C. A schematic diagram of the elements of secondary structure of the native state of CTL9 is shown at the top of each panel, with solid bars, hashed bars, and open arrows indicating alpha-helix, 3–10 helix and beta-strand structure, respectively. The color coding corresponds to that used in Figure 1.

Figure 4

SSP analysis of wild-type CTL9 () and I98A CTL9 () at pH 5.7, 12 °C. A schematic diagram of the elements of secondary structure of the native state of CTL9 is shown at the top of each panel.

Figure 5

Residual dipolar couplings for CTL9 and the I98A mutant. Measurements were made at pH 5.7, 12 °C. (A) Plot of D_NH for the wild-type CTL9 versus residue numner. (B) Plot of D_NH (black bars) and SSP scores () for I98A CTL9 versus residue number. A schematic diagram of the elements of secondary structure of the native state of CTL9 is shown at the top of each panel.

Figure 6

Summary of NOEs observed for I98A CTL9 at pH 5.7, 12 °C. (A) Plot of the volume of the sequential amide proton NOEs versus residue number. (B) Plot of normalized sequential amide proton NOEs versus residue numner. Peak volumes are normalized as the ratio of observed d_NN(i,i+1) NOE crosspeaks to the diagonal peaks.

Figure 7

Plots of ¹⁵N R₂ relaxation rates for wild-type CTL9 () and the cold denatured state of I98A CTL9 (). The black solid line (−) is the best fit to the phenomenological model of Schwalbe and coworkers. Data was collected at 12°C for both proteins.

Figure 8

Comparison of the SSP scores determined for the pH 3.8 unfolded state of wild type CTL9 with those determined for the cold denatured state of I98A CTL9. The SSP scores for the pH 3.8 unfolded state were previously reported (60).