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Stress response in eukarya: Structural studies on eukaryotic heat shock transcription factors
Heat shock proteins are produced as a response to cellular stress. Commonly they seem to function as molecular chaperones by stabilizing cellular proteins and preventing aggregation. Several heat shock proteins have been described in a number of organisms ranging from bacteria to human. The expression of heat shock proteins is under the control of a number of evolutionarily well conserved heat shock transcription factors (HSFs) in eukarya. Presently three HSF’s have been identified in mouse and human; HSF-1, HSF-2 and HSF-4, which are homologous to each other. The amino acid sequence of HSFs from N- to C-terminal ends is composed of a DNA-binding domain (DBD), a coiled-coil trimerisation domain, a regulatory domain and a transcriptional activation domain. The various heat shock transcription factors are thought to regulate distinct genes and be differentially activated. To date the best known eukaryotic heat shock transcription factor is HSF-1, which is activated in response to several kinds of chemical, physical and physiological stresses and also plays other roles in regulating cell growth. HSF-1 knock-out mice are extremely sensitive to thermal stress, and further give rise to developmental defects and infertile females.
The heat shock response in eukarya is highly cooperative and strictly regulated with the involvement of many cellular factors. The stress-induced activation on HSFs can trigger a series of biochemical events in the cell, including the trimerization of HSF-1, translocation from cytoplasm into the nucleus and the formation of nuclear granules. Many details of the mechanism are still scarce. Structural studies can undoubtedly benefit in deep insight to this complex cellular procedure. Up till now, however, only the structure of the DNA-binding domain of HSF-1 has been determined by X-ray crystallography, which reveals a winged helix (wH) DNA-binding motif. Structure information for the remainder of HSFs is still lacking, probably because of the difficulty in protein expression, purification or crystallization. Therefore, the main purpose in this study is to solve these problems and obtain single crystals of the full-length proteins. Two constructs have been made to express full-length murine HSF-1 and HSF-2, but both expression and purification need to be optimized to give good purity and sufficient yield for crystallographic studies.
Ex-jobs provided for students
1. Optimize both the expression and purification protocol to achieve the maximal yield and best purity of murine HSF-1 and HSF-2, and probably human HSF-1 or HSF-2.
2. Produce the above protein till sufficient amount for crystallization.
3. (Probably) Crystallization and/or collection of the diffraction data dependent on the progress of the project
The students will have the chance to be trained with the following experimental skills
• Bacterial culture, cell lysis and protein electrophoresis
• The operation of the ÄKTA system for protein purification. They will become familiar with various chromatographic techniques including affinity chromatography and gel filtration.
• (Probably) make the first step in the field of protein crystallography and structural biology
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