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Thermal adaptation of the ribosomal chaperone trigger factor

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Amandine Godin, Philipp Schmidpeter*, Franz X. Schmid*, Georges Feller

Laboratory of Biochemistry, Centre for Protein Engineering, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium *Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany

Introduction

Trigger factor (TF) is a 48 kDa protein involved in folding in bacteria. It is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. Proteomics studies have recently revealed that the trigger factor is the main upregulated protein in the psychrophilic microorganism Pseudoalteromonas

haloplanktis grown at 4°C (compared to 18°C). Moreover, the expression of the two

other major bacterial chaperones, DnaK and GroEL, is downregulated at 4°C. Therefore, phTF seems to play a key role in cold adaptation of the Antarctic bacterium. ribosome trigger factor (TF) native native native DnaJ DnaK (+GrpE, ATP) GroEL (+ ATP) GroES

E. coli trigger factor

N -term domai n C -term domai n PPIas e “head” “arm1” “arm2” “tail” “ribosome binding”

Comparative study of three trigger factors from different

thermal origins

Chaperone activity

Protein Source Estimated environmental T°

PhTF Pseudoalteromonas haloplanktis TAC125 < 0°C (psychrophilic)

EcTF Escherichia coli RR1 37°C (mesophilic)

TmTF Thermotoga maritima DSM3109 85-90°C (hyperthermophilic)

In order to study the adaptation of this chaperone to temperature, we have produced and purified three different TFs, and we have investigated their chaperone function, PPIase activity and interaction with unfolded substrates.

[TF]/[GAPDH] 0,0 0,5 1,0 1,5 2,0 2,5 3,0 A g g reg at io n ( % ) 0 20 40 60 80 100 PhTF 20°C PhTF 15°C EcTF 20°C TmTF 20°C

Effect on the aggregation of GdmCl-denatured GAPDH, induced upon dilution:

EcTF and TmTF - Gradual prevention of aggregation at 20°C

PhTF (Tm=33°C) - No protection against aggregation at 20°C,

but chaperone activity detected at 15°C after cold incubation [TF]/[GAPDH] 0 1 2 3 4 5 6 7 8 9 10 G A PD H act iv it y r eco v ery ( % ) 0 5 10 15 20 25 30 Prevention of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) aggregation by TFs

Reactivation of D-glyceraldehyde-3-phosphate dehydrogenase by TFs

EcTF 15°C

PhTF

TmTF

Effect on the reactivation of GdmCl-denatured GAPDH: PhTF - Slight improvement

EcTF - Enhancement at low TF concentration, inhibition at

high concentration TmTF - Inhibition 0.0 0.5 1.0 1.5 -14.00 -12.00 -10.00 -8.00 -6.00 -4.00 -2.00 0.00 -0.60 -0.50 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0 10 20 30 40 50 60 70 Time (min) µcal /sec Molar Ratio kcal m ol -1 of inj ectant 0.0 0.5 1.0 -6.00 -4.00 -2.00 0.00 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0 10 20 30 40 50 60 70 Time (min) µcal /sec Molar Ratio kcal m ol -1 of inj ect an t

Interaction EcTF-α-casein Interaction TmTF-α-casein

PPIase activity

EcTF

- PPIase activity detected

- Lower specifity towards peptides having a charged residue before P

PhTF

- PPIase activity detected

- Less active than EcTF, even at 15°C - Likely similar specifity

TmTF

- Almost inactive

Tetrapeptide assay Green Fluorescent Protein mutant 2 refolding assay

in the presence of TFs Time (sec) 100 200 300 400 500 600 700 800 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Time (sec) 100 200 300 400 500 600 700 800 G FP Fluo res cence ( % ) 0 10 20 30 40 50 60 70 80 90 100 110 120 130 EcTF EcTF+GroELS PhTF+GroELS PhTF GroELS No chaperone TmTF+GroELS GroELS No chaperone TmTF + GroELS - GroELS 15°C

Cooperation with the chaperonin GroEL/ES+ATP: GroELS

- Improvement of GFP refolding yield and acceleration of the first phase of the kinetics

EcTF+GroELS

- Competition between the 2 chaperones

PhTF+GroELS

- Slight improvement of the yield

TmTF+GroELS

- Cooperative effect

- Prior incubation with TmTF is favorable to the refolding

- EcTF and TmTF: Binding to an unfolded protein,

and not necessary to a refolding protein

- No interaction detected with PhTF

- Stopped-flow affinity kinetics: high affinity of

TmTF for RCM-RNase T1 Effect on the refolding of acid-denatured GFP:

PhTF - Slight improvement

EcTF - Enhancement at low TF concentration

TmTF - Inhibition

 Consistent with GAPDH reactivation assay

 Also performed at physiological temperatures of

each TFs: results similar to those obtained at 15°C

Interaction TFs-unfolded substrates

Isothermal Titration Calorimetry

15°C 15°C

Conclusions

This characterization of extremophilic TFs, performed under identical experimental conditions for the three chaperones, suggests that the trigger factor possesses thermal adaptations related to the strain lifestyle.

 Thermophilic TF behaves as a holdase, forming a complex with proteins to protect them at high physiological temperatures prone to protein aggregation before their folding by other chaperone systems.

Psychrophilic TF promotes the folding at any concentration and acts as a “true” chaperone. The lower efficiency of PhTF may be caused by the experimental conditions which

are far from P. haloplanktis’ life conditions. It is also possible that PhTF acts only during polypeptides synthesis at the ribosome, as very low temperatures prevent aggregation.

Mesophilic TF seems to be versatile, with a characteristic holding-chaperone activity.

This fundamental study could have potential applications in biomedical field (diseases due to protein aggregation such as Alzheimer), and for protein expression systems in biotech industry.

Ferbitz et al., Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature, 2004. 431(7008): p. 590-6.

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