Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Abstract

Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein⅐peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 M sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4°C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 M sarcosine increases with temperature, ranging from ؊0.52 kcal mol ؊1 at 20°C to ؊5.4 kcal mol ؊1 at 60°C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.Osmolytes are molecules used in nature to protect organisms against stresses of high osmotic pressure. These compounds have also been found to stabilize the native state of proteins relative to the unfolded state. The mechanism of this stabilization is not completely understood (1-4), although it is believed to result primarily from an unfavorable free energy of interaction between the osmolyte and the unfolded state of the protein (5, 6). Proteins retain activity in the presence of osmolytes suggesting that native state structure and dynamics are not greatly perturbed. However, there is little high resolution structural information available for proteins in the presence of osmolytes. The main classes of osmolytes are sugars, methyl ammonium derivatives, polyhydric alcohols, and amino acids and their derivatives (1). Molar concentrations of all the above classes of molecules have been shown to stabilize proteins. Many organisms accumulate osmolytes under conditions of water stress, such as high salinity, desiccation or freezing. In vivo, amino acid derivatives also counteract the accumulation of urea, which is capable of denaturing proteins. Marine cartilaginous fishes use, as osmolytes, a combination of urea and methylamines, i.e. a denaturant and a stabilizer, in a 2:1 ratio (1,7,8). Stability studies on RNase T1, RNase A, and other proteins have shown that, if the 2:1 ratio of urea:methylamine is maintained, the methylamine is able to counteract the destabilizing effect of the denaturant (1,7,8).In an effort to further our understanding of the basis of osmolyte stabilization of proteins, we have studied the stabilization of the fragment complementation system ribonuclease S (RNase S) 1 in four structurally similar osmolytes, namely sarcosine, betaine, trimethylamine-N-oxide (TMAO), and taurine. RNase S is a complex of two fragm...

Counteracting Osmolyte Trimethylamine N-Oxide Destabilizes Proteins at pH below Its pK

“…One of the important conclusions reached from the results presented in Table I is that TMAO stabilizes proteins if pH is Ն5.0 and destabilizes proteins if pH is Ͻ5.0. It is noteworthy that our findings at pH 5.0 and above is consistent with earlier reports (7)(8)(9)(10)(11)36). However, no report is available for the effect of TMAO on protein stability at pH values of Ͻ5.0.…”

“…It has been reported that the structure of the native and denatured states of proteins are not affected in the presence of TMAO (7,26). The results shown in Fig.…”

“…The effect of TMAO on protein stability and enzyme activity has been widely studied. This osmolyte has been shown in vitro to do the following: (i) increase the melting temperature as well as the unfolding free energy of proteins (7)(8)(9)(10)(11); (ii) offset the destabilizing effects of urea (8,10,11); (iii) restore the enzyme activity that is lost upon urea treatment (12,13); (iv) force the folding of unstructured proteins (4,(12)(13)(14)(15); (v) favor the protein self-association and polymerization of microtubules (16 -18); (vi) correct temperature-sensitive folding defects (19); and (vii) interfere with the formation of scrape prion protein (20). TMAO has been shown in vivo to counteract the damaging effects of salts (21), hydrostatic pressure (22,23), and urea (24,25) on proteins.…”

Thermodynamics and mechanism of cutinase stabilization by trehalose

“…Most of the studies have neglected the possibility that the presence of osmolytes would change the protein structure, especially the unfolded state as indicated here and in other studies. 12,[33][34][35]37,42,50 Reasoning according to the new view of protein folding based on funnels or energy landscapes, 51 the conformational space accessible to the unfolded state decreases in the presence of osmolytes when the unfolded state acquires residual native interactions that channel the folding of the protein. Osmolytes may channel folding along particular pathways by preferentially stabilizing one or more structural components of an ensemble.…”

Naturally Occurring Osmolytes Modulate the Nanomechanical Properties of Polycystic Kidney Disease Domains

“…For example, cartilaginous fish concentrate trimethylamine N-oxide (TMAO), to offset the damaging effects of urea on protein function, and it is one of the best studied protecting osmolytes (e.g. 16 -20).The mechanisms by which protecting osmolytes promote protein folding, increase protein stability, and induce conformational changes have been the focus of intense investigation (15,18,19,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). However, nothing is known about the effects of denaturing or protecting osmolytes on the mechanical properties of PKD domains.…”