Molecular Adaptation Strategies to High Temperature and Thermal Denaturation Mechanism of the D-Trehalose/D-Maltose-Binding Protein From the Hyperthermophilic Archaeon Thermococcus litoralis Dimitrios Fessas, 1 Maria Staiano, 2 Alberto Barbiroli, 1 Anna Marabotti, 3 Alberto Schiraldi, 1 Antonio Varriale, 2 Mose’ Rossi, 2 and Sabato D’Auria 2 * 1 Department of Food Science, Technology and Microbiology, University of Milan, Milan, Italy 2 Istituto di Biochimica delle Proteine, CNR, Napoli, Italy 3 Laboratorio di Bioinformatica, Istituto di Scienze dell’Alimentazione, CNR, Avellino, Italy ABSTRACT The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose uptake system. In the hyperthermophilic archaeon T. litor- alis this is mediated by a protein-dependent ATP- binding cassette system transporter. The gene cod- ing for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombi- nant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity. In this work we used dif- ferential scanning calorimetry studies together with a detailed analysis, at the molecular level, of the three-dimensional protein structure to shed light on the basis of the high thermostability exhib- ited by the recombinant TMBP from the archaeon T. litoralis. The obtained data suggest that the pres- ence of trehalose does not change the overall mech- anism of the denaturation of this protein but it selectively modifies the stability of the TMBP struc- tural domains. Proteins 2007;67:1002–1009. V V C 2007 Wiley-Liss, Inc. Key words: trehalose/maltose-binding protein; peri- plasmic protein; differential scanning calorimetry; archaeon INTRODUCTION The general interest in biomolecules isolated from ther- mophilic organisms was originally due to the biotechnolog- ical advantages offered by the utilization of these high sta- ble molecules in industrial processes. 1 In fact enzymes and proteins isolated from thermophilic microorganisms exhibit a high stability in conditions usually used to dena- ture proteins: high temperature, ionic strength, extreme pH values, elevated concentration of detergents, and cha- otropic agents. 2 In addition to the potential industrial applications, it is important to highlight that proteins and enzymes that are stable and active over 1008C represent good models to shed light on the molecular adaptation of life at high temperature. 3 The D-trehalose/D-maltose-binding protein (TMBP) is one component of the trehalose (Tre) and maltose (Mal) uptake system, which in the hyperthermophilic archaeon Thermococcus litoralis, is mediated by a protein-depend- ent ATP-binding cassette system transporter. 4 TMBP from T. litoralis is a monomeric 48 kDa two-domain mac- romolecule containing 12 tryptophan residues. 5 TMBP shares common structural motifs with a number of other sugar-binding proteins. This class of biomolecules is com- posed of proteins whose structure consists of two globular domains connected by a hinge region made of two or three short peptide segments. The two domains are formed by noncontiguous polypeptide stretches and exhibit similar tertiary structure. The ligand-binding site is located in the deep cleft between the two domains, and the binding is accompanied by a movement of the two lobes as well as by conformational changes in the hinge region. 6 The gene coding for the thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. 7 The recombinant TMBP has been purified to homogeneity and character- ized. It exhibits the same functional and structural prop- erties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity (K d of 0.160 lM). 7 In a recent work, 8 we have investigated the structure and the dynamics of the recombinant TMBP by Fourier Abbreviations: DSC, differential scanning calorimetry; TMBP, D- trehalose/D-maltose-binding protein; TMBP/Tre, TMBP in the pres- ence of D-trehalose; Tre, D-trehalose. Grant sponsor: Fondazione Parco Biomedico S. Raffaele; Grant number: 1/014/06/0; Grant sponsor: Ministero degli Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale. *Correspondence to: Dr. Sabato D’Auria, Institute of Protein Bio- chemistry, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy. E-mail: s.dauria@ibp.cnr.it Received 28 June 2006; Revised 15 November 2006; Accepted 20 December 2006 Published online 20 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21383 V V C 2007 WILEY-LISS, INC. PROTEINS: Structure, Function, and Bioinformatics 67:1002–1009 (2007)