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.

Observation of Two Families of Folding Pathways of BBL

“…2 B) approaching the native-state basin. We observe side-chain van der Waals contacts between the N-and Cterminals, most importantly between Ser 3 and Pro 4 at the N-terminal and Leu 38 , Ala 39 , and Lys 40 at the C-terminal, but no hydrogen bonding is seen. From Steps 15 to 16 of Path I, the end-to-end distance sharply rises to above the native value.…”

“…Biophysical Journal 100(10) 2457-2465 coexist under the same conditions. Recent experimental and computational studies have shown that pH and salt concentrations can modulate the folding cooperativity of BBL (39) and the native structure changes with solvent conditions (40). These observations imply that the (free) energy landscape varies with solvent conditions.…”

Multiscaled exploration of coupled folding and binding of an intrinsically disordered molecular recognition element in measles virus nucleoprotein

“…In recent years, an increasing number of studies emphasized a relationship between MG and downhill folding (67,91,92), between downhill folding and IDP (12,65,90), and finally between MG and IDP (4, 26, 88, 89, 93, 94). From the energy landscape perspective, the MG state is between the unfolded state at the top of the funnel and the folded state at the bottom of the funnel (78).…”

The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein

“…One of the most relevant features of surface charge-charge interactions is the Debye-Hückel screening exerted by salt ions that accumulate around surface charges thus strongly weakening electrostatic forces between ionizable residues of the protein (either attractive or repulsive interactions). Accordingly, protein thermodynamic and kinetic stabilities [21] as well as protein folding barriers [26] can be efficiently modulated through electrostatic screening of surface charges. Salt effects on protein stability vary according to their concentration (i.e.…”

“…At low ionic strength (typically below 0.2 M of monovalent salt), salt ions may interact with surface charges of opposite sign causing changes in protein stability that exponentially depend on the ionic strength, even though other mechanisms such as ion specific binding may also be involved. At high ionic strength, salt ions may affect protein stability by perturbing the hydration shell of the protein (which is ion dependent, according to the Hofmeister series) and this effect scales linearly with salt concentration [21, 26, 27]. …”