ARTICLE A. Budi Æ S. Legge Æ H. Treutlein Æ I. Yarovsky Effect of external stresses on protein conformation: a computer modelling study Received: 13 March 2003 / Revised: 27 August 2003 / Accepted: 28 August 2003 / Published online: 23 October 2003 Ó EBSA 2003 Abstract The increasing use of digital technologies such as mobile phones has led to major health concerns about the effects of non-ionizing pulsed radiation exposure. We believe that the health implications of exposure to radia- tion cannot be fully understood without establishing the molecular mechanisms of biological effects of pulsed microwaves. We aim to establish methods for studying the molecular mechanisms of protein structural and energetic changes occurring due to external stresses related to non- ionizing radiation by using a combination of experimental and theoretical approaches. In this paper, we present the results from our fully atomistic simulation study of chemical and thermal stress response of a prototype protein, insulin. We performed a series of molecular dynamics simulations of insulin in solution under equi- librium conditions, under chemical stress (imitated by reducing the disulfide bonds in the protein molecule), and under short-lived thermal stress (imitated by increasing simulation temperature for up to 2 ns). The resultant protein conformational behaviour was analysed for var- ious properties with the aim of establishing analysis rou- tines for classification of protein unfolding pathways and associated molecular mechanisms. Keywords Conformational analysis Æ Insulin Æ Molecular dynamics simulations Æ Non-ionizing radiation Æ Protein unfolding Introduction The function of a protein is intrinsically associated with its three-dimensional conformation. Therefore, any changes in the geometry of the protein, particularly in the region of the active site, have the potential to alter its biological function. Previous experiments have shown that there are links between external stress, such as electromagnetic fields, and protein structure. In several reported cases, the presence of weak microwave fields was enough to induce physiological changes in organ- isms (de Pomerai et al. 2002; French et al. 2000). Al- though the use of electromagnetic fields as a stressor has been investigated (Bohr and Bohr 2000; de Pomerai et al. 2002; French et al. 1997), these experimental studies were done via directly observable tissue damage, i.e. at long experimental timescales and relatively large length (size) scales. Therefore, the exact cause of these physiological changes, and the extent of the contribution of thermal and/or chemical stress to these changes, are not fully understood. It is now believed that the effect of pulsed microwave radiation may only become apparent at short timescales (within the nanosecond region) and at the microscopic length scale, i.e. at the molecular level (Laurence et al. 2000). Therefore, it may not be possible to apply tradi- tional experimental measurements to identify such short-lived microscopic effects which may cause identi- fiable physiological changes. Conformational changes in proteins trigger the expression of small heat shock response proteins (sHSPs) as part of a defence mechanism used by the cell. The sHSPs are molecular chaperones that either assist in the refolding of the protein or, alternatively, in the degradation of the protein. The expression of sHSPs is not simply a response to heat, but is a general response to other stressors such as alcohol, heavy metals, oxida- tion, and osmotic pressure changes. The over-expression of sHSPs has been associated with increased oncogenesis and metastasis, as well as increased resistance to Submitted as a record of the 2002 Australian Biophysical Society meeting A. Budi Æ S. Legge Æ I. Yarovsky (&) Department of Applied Physics, RMIT University, GPO Box 2476V, 3001 Melbourne, Victoria, Australia E-mail: irene.yarovsky@rmit.edu.au Tel.: +61-3-99252571 Fax: +61-3-99255290 H. Treutlein Cytopia Pty. Ltd., Level 5, Baker Institute, Commercial Road, 3004 Melbourne, Victoria, Australia Eur Biophys J (2004) 33: 121–129 DOI 10.1007/s00249-003-0359-y