1. Heat shock Proteins (HSPs) Heat shock proteins (HSP) are expressed in response to various biological stresses, including heat, high pressures, and toxic compounds. It is also one of the most abundant cellular proteins found under non- stress conditions Hsp90 is part of a family of proteins known as "chaperones," which are solely dedicated to helping other proteins fold and assume their proper functions. The chaperones Hsp70 and Hsp90 together with co-chaperones function to fold proteins in the cytoplasm. Sometimes Hsp70 and Hsp90 function sequentially to fold the same protein Cells are vigilant about getting these folds right because misfolded proteins can change the normal life of the cell. In some cases change is good, in others deadly. When HSP90 is compromised the number of morphological changes increases, which lead to formation of inactive or abnormally active polypeptides. Domain structure of HSP90.

2. Mad Cows, People & yeast What do "mad cows," people with neurodegenerative diseases and an unusual type of yeast have in common? They are all experiencing the effects of misfolded proteins, according to HHMI investigator Susan Lindquist of the University of Chicago. Her research identified a role for HSP90 in the process of evolution. They have reported that fruit flies that make too little of the Hsp90 protein develop dramatic deformities, such as an extra antenna, additional bristles, notched wings or malformed eyes. The defects result from multiple hidden variations in the genome. When affected flies are interbred, these factors are enriched and subsequent generations have the same deformities, even though they have normal levels of Hsp90. Susan Lindquist University of Chicago "My view is that molecular chaperones are a way of changing the traits of an organism that arose very early in evolution. They might be as old as RNA and DNA." Source:http://www.hhmi.org/annual98/research/madcow.htmlhttp://www.hhmi.org/annual98/research/madcow.html

3. HSPs in protein folding The diagram shows the role of heat- shock proteins and a chaperonin in protein folding. As the ribosome moves along the molecule of messenger RNA, a chain of amino acids is built up to form a new protein molecule. The chain is protected against unwanted interactions with other cytoplasmic molecules by heat-shock proteins and a chaperonin molecule until it has successfully completed its folding. Source: (http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/HSP/FUNCTION.HTML)

4. HSP90-alpha protein sequence HSP90alpha 732 aa HSP90beta 724 aa >HSP90alpha MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFME ALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAE EKEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKKKKKKIKEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFDL FENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLPLNISREMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSAS GDEMVSLKDYCTRMKENQKHIYYITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLELPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVEKV VVSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDP TADDTSAAVTEEMPPLEGDDDTSRMEEVD Green bases = Geldanamycin-Binding Domain Brown bases = Transmembrane segments as predicted by Tmap. Hydrophobic: A,C, I,L, M, F, V,P, Y,W Hydrophilic: R,N, H,D, E,Q, K, S,T Either G GREASE output TMAP output

5. BLASTp With the HSP90 sequence in hand we used Blastp to find homologous sequences We were surprised to find a lot of homologous sequences across many species like Humans, Chicken,Pig, Mouse,Horse,Fish, Coral,fruit fly, mosquito, nematode,& even crops like rice, maize & tobacco. The first 100 matches had e-values ranging from 0 to e-153, so they were *very* strong matches indicating a high degree of conservation of the protein through evolution. IDNameScoreEvalue 304882heat shock 90kDa protein 1, alpha [Homo sapiens] N... 12470.01247 352285heat shock protein 1, alpha [Mus musculus] NP_0346... 8250.0825 761972heat shock protein 86 [Rattus norvegicus] NP_78693... 8250.0825 341493heat shock protein 90A [Cricetulus griseus] AAA369... 8170.0817 609431heat shock protein 90 - chicken 8160.0816 609432heat shock protein 84 - mouse 7450.0745 449511(Q9W6K6) Heat shock protein hsp90 beta [Salmo sala... 7310.0731 459017heat shock protein hsp90 [Oncorhynchus tshawytscha... 7300.0730 446434heat shock protein hsp90beta [Danio rerio] AAC2156... 7290.0729 361999heat shock protein 90 [Rattus sp.] AAB23369.1 [S45... 7240.0724 460597heat shock protein 90 [Pleurodeles waltl] AAA92343... 7190.0719 73860490-kDa heat shock protein [Bombyx mori] BAB41209.1... 7120.0712 146263Heat shock protein 83 CG1242-PA [Drosophila melano... 6690.0669 755572heat shock protein 90 [Dendronephthya klunzingeri]... 6620.0662 226533(P33126) Heat shock protein 82 [Oryza sativa (Rice)] 612e-174 1888761heat shock protein 82 - common tobacco (fragment) 612e-174 252633heat shock protein [Arabidopsis thaliana] CAA72513... 600e-170 236351(Q9XGF1) HSP80-2 [Triticum aestivum (Wheat)] 598e-169 283559(Q08277) Heat shock protein 82 [Zea mays (Maize)] 593e-168 152674heat shock protein 86 [Plasmodium falciparum] AAA6... 591e-167 1899880(Q8LLI6) Heat shock protein Hsp90 [Achlya ambisex... 579e-164 245912heat shock protein 90 [Lycopersicon esculentum] AA... 544e-153

6. Multiple sequence alignment Multiple sequence alignments were done using ClustalW using different species & the following unrooted phylogenetic tree was generated.

7. Rooted phylogenetic tree.

8. Structure analysis Comparison between Open & Closed conformations of human HSP90 alpha Transmembrane segments in yellow We found ‘open’ and ‘closed’ conformations for the Geldanamycin- Binding Domain of the Human Hsp90 protein & decided to study their differences. The parts in yellow are the selected residues which are also the transmembrane segments. The residues ‘gtia’ in the sequence viewer show where the structures differ. The same region is depicted as the little grey segment at one end of the transmembrane segments

9. Comparison of HSP90 structures in Yeast & Human We compared the HSP90 structures in yeast & human & found that the protein structures were very similar. The picture below shows the 2 structures superimposed with the highlighted portion showing an additional residue in the yeast sequence. Structures compared: 1YES Human Hsp90 Geldanamycin-Binding Domain, "open" Conformation [mmdbId:7483]1YES 1AMW1AMW : Atp Binding Site In The Hsp90 Molecular Chaperone,Saccharomyces cerevisiae (yeast) [mmdbId:7950]Saccharomyces cerevisiae Red : identical residues Blue : similar residues Yellow: selected residues

10. Microarray Data We found microarray data on the Cancer Genome Anatomy Project web site http://cgap.nci.nih.gov/Genes/GeneFinder HSP90-alpha This screen shot shows the microarray data for HSP90 alpha expression in various types of cancers.

11. HSP90-beta This screen shot shows the microarray data for HSP90 beta expression in various types of cancers.

12. Current Studies on HSP90 Changes in protein conformation are involved in some of the most devastating and intractable diseases. “Studies in yeast may help us decipher the fundamental nature of these disorders, including Creutzfeldt-Jakob, Alzheimer’s, Huntington’s, and Parkinson’s disease in humans and mad cow disease in cattle. Several of the protein culprits are being imported into yeast, which allows for the manipulation & study of their folding transitions & testing of therapeutic strategies. (http://www.wi.mit.edu/nap/pdfs/Directors_Report/dir_lindquist02.pdf) Conclusion: HSP90 is a powerful evolutionary mechanism that ensures apparent genetic stability at physiological conditions and at the same time allows the mutations that could rapidly become manifest under stress. References : http://www.stanford.edu/class/gene211/hsp90_search http://www.chemie.tu-muenchen.de/biotech/en/hsp90-e.html http://www.hhmi.org/annual98/research/madcow.html www.ashland.edu/~kstine/Research/Stress%20proteins.pdf