Investigation of potential EMF induced conformational changes of thermosensor protein GrpE of E. Coli in real time

Abstract

Previous studies on potential interactions of electromagnetic field (EMF) with proteins have reported that EMFs affect protein structure and folding kinetics. In this study the isolated thermosensor protein GrpE of the Hsp70 chaperone system of E. coli was exposed to EMFs at different frequencies and field strengths under strictly controlled environmental condition. Potential structural changes were monitored with circular dichroism spectroscopy whilst simultaneously recording the temperature at the point of observation. The specific properties of the protein GrpE allow for distinguishing between thermally and non-thermally induced conformational changes. Since the transition is fully reversible, any direct effects of EMF on the conformation will results in a shift of the equilibrium states. The overall uncertainty of the experimental system was evaluated and based on these results the number of experiments needed to achieve a certain detection limit was determined. Potential effects of EMF were investigated in long, medium and immediate reaction time for various field strengths up to 3000 V/m in the protein solution and different signals including GSM. Shifts of the thermal equilibrium corresponding to a temperature increase of only 0.1 K can be detected in minimum. The results obtained so far suggest that the conformational equilibrium of GrpE is insensitive to weak electromagnetic fields. Future experiments will focus on the investigation of a potential influence of EMFs on protein dynamics by assessing shifts in the conformational equilibrium characteristics over an extended temperature range. Introduction Studies on the potential effects of electromagnetic fields (EMF) on different cell types have been examined alterations in complex cellular features and processes, such as genetic damage, proliferation, differentiation, apoptosis and gene expression. Mostly narrow-band signals such as emitted by mobile phones in the GSM or UMTS bands were applied. The biological analyses were carried out after the exposure to EMF. The results remain controversial, and in most cases their replication has proven difficult. Previous studies on single proteins in simple protein-buffer systems have already reported effects of EMF on protein structure and folding kinetics. Since the molecular mechanisms are still unclear, a more mechanismoriented approach is needed. In this study the isolated thermosensor protein GrpE of the Hsp70 chaperone system of Escherichia coli is exposed to EMFs while simultaneously monitoring its potential structural changes via circular dichroism spectroscopy. Thus, the point of observation becomes identical with the potential interaction site in space and time, a situation that will allow for the detection of even small effects of EMFs. By using isolated proteins, the complexity of the target is reduced from the cellular to the molecular level. Heat-shock proteins (Hsp) are an important group of cellular stress response proteins. They act primarily as molecular chaperones to eliminate unfolded or misfolded proteins, which are generated under cellular stress. This stress response can be induced by various external factors, including increased temperature, chemicals, oxidative stress, heavy metals, ionizing and non-ionizing radiation and ultra-fine carbon black particles. An increased expression level of Hsp70 thus confers protection against cellular stress. Direct effects on the conformation of a component such as GrpE of the Hsp70 system of Escherichia coli (DnaK/DnaJ/GrpE) could therefore lead to a change in response to cellular stress. BEYER, JELESAROV, CHRISTEN, FROHLICH Figure 1: On the left side: the increase and decrease of the temperature is shown for the buffer solution applying 3 minutes of EMF exposure; on the right: quantile plot of 50 GrpE CD-measurements. Objective GrpE undergoes a completely reversible conformational change upon an increase in temperature within a temperature range from 8 to 60 °C. Since the transition is fully reversible, a temperature-dependent conformational equilibrium ensues. Any direct effects on the conformation will results in a shift of this equilibrium. In this study the hypothesis on potential direct effects from RF electromagnetic field exposure occurring in everyday situations on the structure of GrpE and its protein folding kinetics shall be investigated. The underlying questions are: Do electromagnetic fields shift the conformational equilibrium of GrpE? What is the observable effect size by the exposure setup used? Furthermore, the threshold magnitude of EMF for a potential effect, as well as the frequency ranges where it occurs can be obtained. Provided that all thermal, electro-magnetical, chemical and optical conditions within irradiated volume are known and controlled this configuration should allow for discrimination of non-thermally induced structural changes from those caused by temperature changes. Once an effect is detected and its thresholds are found, the molecular mechanisms might be explored in detail by genetic engineering of GrpE, e.g. by deleting or introducing positively or negatively charged residues, mutational analysis of the hydrophobic core, etc.