Thermodynamic stability of a cold‐adapted protein, type III antifreeze protein, and energetic contribution of salt bridges

Abstract

A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is twostate, the psychrophilic type III AFP has a much lower thermodynamic stability at 25°C, ;3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0°C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (;1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25°C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0°C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.Keywords: psychrophilic protein; cold adaptation; antifreeze protein; thermodynamic stability; salt bridge Psychrophilic, or cold-adapted, proteins are widespread in organisms living in cold environments. These proteins constitute excellent models to study molecular adaptations to extreme conditions (Jaenicke 1990; Gerday 1997, 2003;Jaenicke and Böhm 1998;Smalas et al. 2000;Arnold et al. 2001;D'Amico et al. 2002;Marx et al. 2004), and may have many biotechnological applications (Feller and Gerday 1997;Russell 1998;Cavicchioli et al. 2002;Marx et al. 2004). Most studies on the stability of cold-adapted proteins (Ushakov 1964;Chen and Berns 1978;Cheng and DeVries 1989; Reprint requests to: Mauricio G. Mateu, Centro de Biología Molecular ''Severo Ochoa'' (UAM-CSIC), Universidad Autó noma de Madrid, Cantoblanco, 28049 Madrid, Spain; e-mail: mgarcia@cbm. uam.es; fax: +34-914974799.Abbreviations: AFP, antifreeze protein; CD, circular dichroism; GdmHCl, guanidinium hydrochloride; T m , transition temperature; DH u Tm , enthalpy of unfolding at the transition temperature; DG u H2O , free energy of unfolding extrapolated to absence of denaturant; m, variation in the free energy of unfolding with the denaturant concentration; DDG u H2O , the change in DG u H2O upon mutation; DDG intXY , the coupling energy between groups X and Y in a protein.Article published online ahead of print. Article and publication date are at

Electro-Optical Properties Characterization of Fish Type III Antifreeze Protein

“…In spite of this, the spectrum of type III AFP shows a native-absorption band between 215 and 230 nm, and it allows us to conduct unfolding experiments. It is important to note that the far UV-CD spectrum results are similar in shape to that obtained by other groups in the presence of salt [17,19]. …”

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

“…By extrapolating the k 2 values obtained at [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] C to the T m at 28.5 C (R 2 5 0.981, exponential regression) we obtain a k 2 and k 1 of 0.00526 min…”

“…A high refolding capacity has also been observed for other AFPs. 13,[19][20][21][22] In two cases the activity of the protein was measured to ensure that the protein refolded to its native state, and only one of these showed that the protein (DAFP-4) had retained most of its activity. Here, we also show that the RmAFP1 has activity almost similar to the untreated control after subjection to 10 heat cycles to both 60 and 70 C. When the protein sample was repeatedly heated to 80 C the DH unfold decreased by 6.5% of the initial value per run (Fig.…”

“…At temperatures above T m (i.e., for K > 1); however, the colloidal stability is essentially governed by k ag . The present knowledge of the stability of AFPs is superficial, and to our knowledge only two experimental studies 12,13 have focused specifically on this topic. However, several observations regarding the thermostabilities of various AFPs have been reported and are presented in Table I.…”

“…Several research groups have reported high refolding abilities of AFPs after heat denaturation, 13,[19][20][21][22] but only in two studies have activity measurements been carried out after an observed refolding to test if the protein had undergone a reverse transition into the native, active form. 19,21 For the P. americanus 17 kDa AFP, it was observed that the activity after being exposed to room temperature (22 C) was not as high as expected on the basis of CD-measurement of refolding.…”