Structural Studies for Rv0352 and Rv3715c By Brian Chiu - PowerPoint PPT Presentation

Structural Studies for Rv0352 and Rv3715c By Brian Chiu

Differential Scanning Fluorimetry Experiments • Purpose: to assess the ability of chemical additives to stabilize target protein • Mainly used for proteins that are soluble but found to have difficulty crystallizing. • The chemical additives may facilitate crystallization of these proteins.

DSF Experiments: How it Works? • Protein is mixed with SYPRO orange dye (and chemical additive) • Fluorescence intensity is measured as temperature rises in 1 o C increment. • As protein gets unfolded, the dye molecules bind to exposed hydrophobic patches and emit fluorescent light.

DSF Experiments : How it Works? Nature Protocols 2 , - 2212 - 2221 (2007)

DSF Experiments: How it Works? • Melting temperature of the protein is calculated through analyzing the change in fluorescence intensity over temperature. • The shift in melting temperature reflects the change in stability of the protein. (Higher melting temperature = More Stable)

Rv0352 • Probable Chaperone Protein DnaJ1 – Hsp40 protein Homolog that regulates activities of Hsp70 chaperone protein through assisting in peptide substrates delivering and controlling ATPase activities involved in folding reaction. • MW = ~42kDa • Cloned into pET22b and expressed well in BL21-Gold (DE3) • 25 o C Expression Temperature • Lysed in NP40 and eluted in Native Buffer pH 8 • No crystal found in previous study

Rv0352 • DSF screen showed positive for Tris pH 7 buffer • Further DSF experiments that demonstrates stabilizing effect of Tris buffer at pH 7 comparing to Tris buffer at other pH’s.

Rv0352

Rv0352 • Rv0352 protein was purified using appropriate buffers at pH 7 • Crystal screens were set up by Mosquito Robot using Rv0352 protein in native buffer at pH 7 (20 mM Tris, pH 7; 300 nm NaCl and 10% glycerol)

Rv3715c • Probable Recombination Protein RecR – a protein required for RecF pathway of homologous recombinational DNA repair • MW = ~24kDa • Cloned into pET22b and expressed well in BL21-Gold (DE3) • 25 o C Expression Temperature • Lysed and eluted in Native Buffer pH 8 • No crystal found in previous study

Rv3715c • DSF screen showed positive for these groups – Coproporphrin I Dihydrochloride – Buffers (Tricine pH 8, Bis-Tris pH 8, and Bicine pH 9) – Certain amino acids (C, Homo-C, N, D, E, Q, and H) – MgCl 2 , nucleic Acids and nucleic acid deriviatives – Sugar-amine Compounds

Rv3715c • Further DSF experiments test individual chemical’s stabilizing effect on protein. – MgCl 2 , cysteine, and homo-cysteine, were found to stabilize the protein. • More DSF experiments were performed to find the [Chemical] that gives us the largest stabilizing effect.

Rv3715c

Rv3715c

Rv3715c • Preparation of MgCl 2 -added protein In DSF experiment 3 mM MgCl 2 : 10 µM protein => 300 : 1 MgCl 2 -added protein should have: 300 : 1 => 91.2 mM MgCl 2 : 304 µM protein • But precipitation was observed in this MgCl 2 -added protein.

Rv3715c • 332 µM protein with 3 mM MgCl 2 was used • Crystal screens were set up by Mosquito Robot

Future Plan • Check crystal screens periodically • May set up more crystal screens • If positive results are obtained, this technique can be applied on other proteins that also have difficulty in forming crystal.

Acknowledgement Mark Winnie Sum Janet Irina Annie Jeanne Students Tung Lisa