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 1oC 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)

• 25oC 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)

• 25oC 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) – MgCl2, nucleic Acids and nucleic acid deriviatives – Sugar-amine Compounds

Rv3715c

• Further DSF experiments test individual

chemical’s stabilizing effect on protein.

– MgCl2, 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 MgCl2-added protein

In DSF experiment 3 mM MgCl2 : 10 µM protein => 300 : 1 MgCl2-added protein should have: 300 : 1 => 91.2 mM MgCl2 : 304 µM protein

• But precipitation was observed in this

MgCl2-added protein.

Rv3715c

• 332 µM protein with 3 mM MgCl2 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

Jeanne Janet Mark Sum Annie Irina Tung Lisa Winnie Students