102 Intramolecular Forces Density in Mesophilic and Thermophilic Proteins: Amino Acid Clusters Based Study Rukman Hertadi 1) and Minoru Kanehisa 2) 1) Biochemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Bandung 2) Bioinformatics Center, Institute for Chemical Research, Kyoto University, Kyoto e-mail: rukman@chem.itb.ac.id Received 15 September 2007, Accepted 26 October 2007 Abstract Thermostability of (hyper)thermophilic enzymes has been taken as an advantage in industry to enhance biochemical reactions at elevated temperature. Factors responsible for the thermostability in this class of proteins, however, still remain unclear despite the many works that have been done to elucidate such factors by performing various comparative studies to homologous pairs of (hyper)thermophilic and mesophilic proteins. In the current work, we elucidated the factors by comparing intramolecular forces density in tertiary structure of mesophilic and (hyper)thermophilic proteins in terms of the content of various types of amino acid clusters. A graph spectral method was employed to probe the charged, hydrophobic and aromatic clusters in each tertiary structure of all classes of thermophilic proteins used in our study. Our results revealed that mesophilic and (hyper)thermophilic proteins contain similar level of all types of amino clusters, thereby stabilized with similar level of high-density intramolecular forces, but the former contain a higher number of non-cluster residues and less stabilized by electrostatic interactions, thereby more sensitive to heat. Keywords: Thermophilic, Graph spectra, Intramolecular force density, Amino acid cluster Abstrak Termostabilitas enzim-enzim (hiper)termofilik telah banyak dimanfaatkan di industri terutama untuk meningkatkan laju reaksi biokimia pada suhu tinggi. Faktor penyebab termostabilitas enzim termostabil masih belum diketahui dengan jelas hingga sekarang meskipun berbagai penelitian dengan melakukan studi komparatif dari pasangan homolog protein mesofilik dan (hiper)termofilik telah banyak dilakukan. Pada studi kali ini, kami mencoba menyelidiki faktor- faktor tersebut dengan membandingkan kerapatan gaya intramolekul dalam struktur tersier dari protein mesofilik dan (hiper)termofilik dengan cara mengevaluasi kandungan berbagai tipe klaster asam amino yang terdapat di dalam ketiga kelas protein termofilik. Metode spektra graf digunakan untuk menentukan keberadaan klaster asam amino bermuatan, hidrofobik, dan aromatik dalam setiap struktur tersier dari semua kelas protein termofilik. Hasil analisis ditemukan bahwa protein mesofilik dan (hiper)termofilik pada dasarnya mengandung jumlah klaster yang sebanding, dengan kata lain distabilkan oleh gaya intramolekular yang sebanding kekuatannya. Akan tetapi, bila ditinjau dari sisi jumlah asam amino non-klaster, protein mesofilik rata-rata memiliki rasio yang lebih besar dibanding pada protein (hiper)termofilik. Disamping itu protein mesofilik juga kurang distabilkan oleh interaksi elektrostatik. Kedua hal inilah menurut hasil studi ini, yang membuat protein mesofilik menjadi lebih sensitif terhadap suhu. Kata kunci: Termofilik, Spektra graf, Kerapatan gaya intramolekul, Klaster asam amino 1. Introduction Protein stability is an aspect that commonly included in most protein characterization. This is due to the wide variations exhibited by a variety of proteins classes. Besides, this property could not be predicted from mere amino acid sequences inspection until recently. The stability aspect of proteins may be divided into two main categories, namely chemical and physical/structural aspects. The chemical aspect associates the stability to the chemical formula of amino acid side chains; chemical properties of individual amino acids, such as polarity, hydrophobicity, pI, and pKa; and the chemical environment, such as solvent, denaturing agent, and pH of the solution. Intra and intermolecular forces, folding energy, thermostability, and mechanical stability under external stress are classified into the physical aspects of the protein structure characterization. In this report, we focus our characterization to one of the physical characterization of proteins, namely thermostability.