Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9

Abstract

In vitro, replacing KCl by potassium glutamate (KGlu), the E. coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu− in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to other stabilizers (KCl, KF, K2SO4) and destabilizers (GuHCl, GuHSCN). GuHSCN titrations at 20 °C, performed as a function of concentration of KGlu or other salt and monitored by NTL9-fluorescence, are analyzed to obtain r-values quantifying the Hofmeister salt concentration (m3)-dependence of the unfolding equilibrium constant Kobs (r-value = −dlnKobs/dm3 = (1/RT) dΔG°obs/dm3 = m-value/RT). r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model-compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 °C, we determine a KGlu r-value of 3.22 m−1, and K2SO4, KF, KCl, GuHCl and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38 and −3.00 m−1 respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values, and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 °C.

Stabilization of SecA ATPase by the primary cytoplasmic salt of Escherichia coli

“…The above results suggest that other proteins within the cytoplasm might also be stabilized by primary cytoplasmic salts, as suggested by studies of the NTL9 ribosomal protein domain . A question of particular interest, of course, is how Glu − might affect the cytoplasmic stability of proteins destined for secretion.…”

“…Together, these data indicate that potassium glutamate (KGlu) is the primary cytoplasmic salt in E. coli . Furthermore, we learned that KGlu can stabilize soluble proteins, because Glu − interacts unfavorably with apolar side‐chains and amide groups . Given these facts, we examined the stability of SecA in KGlu solutions.…”

Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

“…However, this type of interaction is precluded in the (SSB) 65 mode since all ssDNA binding sites are occupied by DNA. We therefore suggest that high cooperativity is promoted via direct interactions between the SSB IDLs as depicted in Figure 6 and that these interactions are promoted by anions such as glutamate and acetate that are preferentially excluded from protein regions [36, 37]. Direct interactions between similar types of low complexity intrinsically disordered regions are believed to drive liquid-liquid phase separation, as exemplified by the LAF-1 protein [55].…”

“…Recent work from the Record lab [36, 37] has shown that glutamate has unfavorable interactions with and is thus preferentially excluded from all hydrocarbon groups, and carboxylate and amide oxygens, but interacts favorably with positively charged amino acids. As a result, KGlu generally promotes folding and assembly processes more than KCl.…”

“…However, we show here that highly cooperative binding can also occur in the (SSB) 65 mode. Cooperative binding still requires the IDL, but is promoted, even at high physiological salt concentrations, by replacing chloride with glutamate or acetate, anions that are preferentially excluded from amide protein surfaces [36, 37]. These results suggest that direct interactions between the IDL regions of SSB tetramers drive cooperativity.…”