The effect of electrostatics on the marginal cooperativity of an ultrafast folding protein

Abstract

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103-108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity.

FIGURE 1.

Salt-induced refolding of protonated BBL measured by far-UV CD. A, circles indicate mean residue ellipticity at 222 nm (α-helical signal) at pH 3 with varying concentrations of LiCl. Red lines are phenomenological fits to sigmoidal curves (i.e. Equation 1) to guide the eye. B, plot of the midpoint temperature as a function of LiCl concentration: (light blue circles and left scale) pH 3, (red circles and right scale) pH 7. C, BBL α-helix signal at 222 nm, pH 3, and 268 K as a function of salt concentration: circles, LiCl; triangles, NaCl; and inverted triangles, CsCl. The black line is a linear fit to the composite data from 1 to 6 m to guide the eye. The red line indicates the CD signal of native BBL at pH 7 and 268 K. Inset, far-UV CD spectra at 268 K for pH 7 (red), pH 3 plus 1 m LiCl (blue), and pH 3 plus 5.3 m LiCl (green).

FIGURE 2.

Structural analysis of the salt-refolded state of BBL by NMR. A, H¹-N¹⁵ SOFAST-HMQC spectra measured at 283 K with ¹⁵N natural abundance for native BBL at pH 7 (red), native BBL at pH 3 and 2 m LiCl (light blue), and acid-denatured BBL at pH 3 (dark blue). The labels indicate some cross-peaks that are characteristic of the BBL native state. B, Hα conformational shifts calculated as the difference between the chemical shift of the protein and the typical random coil values for the same residues. Light blue, data at pH 3 plus 2 m LiCl; red, data at pH 7. C, the aliphatic region in the one-dimensional proton NMR spectrum of BBL at pH 3 with 2 m LiCl (light blue) and without (dark blue). The upper and lower panels on the right show fragments of the NOESY spectrum of BBL at 283 K, pH 3, and 2 m LiCl. NOEs between side chain protons of hydrophobic residues indicating critical tertiary contacts in the native structure of BBL are marked in boxes. The residues of BBL involved in these contacts are as follows: Leu¹⁰, His¹³, Leu¹⁵, Ala¹⁷, Ile²⁰, Leu²⁹, Val³⁴, His³⁷, and Leu³⁸.

FIGURE 3.

Comparing the thermal unfolding of salt-stabilized BBL at pH 3 and native BBL at pH 7. A, first derivative of the CD thermal unfolding curves of BBL at pH 7 (red circles) compared with those at pH 3 and 2 m of LiCl (blue circles), NaCl (blue triangles), and CsCl (blue inverted triangles). Black curves connect the data points to guide the eye. B, CD signal at 222 nm of the BBL high temperature (368 K) denatured state at pH 3 and 2 m LiCl, NaCl, and CsCl. Symbols are as described in A. The red line corresponds to the CD signal at pH 7 and the same temperature. The black line is a fit to an exponential function to guide the eye. Inset, far-UV CD spectra of BBL at 363 K and pH 7 (red), pH 3 plus 1 m LiCl (blue), or pH 3 plus 5 m LiCl (green).

FIGURE 4.

Salt-induced refolding of BBL is independent of its ionization status. A, CD thermal unfolding curves of BBL in the presence of 4 m LiCl and at pH 3 (blue) and pH 7 (orange). Red circles correspond to the thermal unfolding curve at pH 7 without salt. B, first derivates of the thermal unfolding curves shown in A (same color scheme). Black curves connect the data points to guide the eye in both panels.

FIGURE 5.

Ultrafast relaxation kinetics of BBL measured by the nanosecond IR temperature-jump method. A, normalized time-dependent changes in the amide I band absorbance at 1632 cm⁻¹ (ΔABS) after temperature-jumps of ∼10 K to a final temperature of 325 K. Red, decay at pH 7 without salt; orange, decay at pH 7 plus 4 m LiCl; blue, decay at pH 3 plus 4 m LiCl. The black lines show the fits to a single exponential function. B, relaxation rates as a function of temperature for the three experimental conditions of A (color scheme as described in A). The data points shown at pH 7 (red) are the average of three measurements, whereas both data sets at 4 m LiCl correspond to single measurements.