Understanding protein folding with thermal shift assays

How to better understand protein properties in solution using thermal shift assay ?

What does a protein look like ?Most of proteins follow a foldingpathway in order to adopt theirfolding.Hydrophobic regions are in the coreof the protein not exposed to thesolvant.The activity of a protein is due to itsfolding.

Why thermal shift assay (TSA)? For the characterization of our recombinant proteins, we need to have information about folding of our protein. Our choice : Fluorescence-based TSA Small High Quick Easy to use device Throughput Once the program Less than 2 hours Size of a real 96 well PCR to obtain resultstime PCR machine created, just push plate button to do the for one 96 well with a computer assay PCR plate

Principle of TSA• SyproOrange fluorescence (excitationat 470 nm/ emission at 570 nm) isquenched in an aqueous environment• As the temperature rises, the proteinundergoes thermal unfolding andexposes its hydrophobic core region• SyproOrange then binds tohydrophobic regions andbecomes unquenched• Fluorescence is plotted andmonitored against temperature• The midpoint of the proteinunfolding transition is defined asthe Tm (melting temperature)• The first derivative is calculated tomore accurately determine theinflection point and thus the Tm

How to do TSA on CFX 96 ?Sample : 2.5 µg of purified protein in 23 µl tested with 2 µl of Sypro Orange (25µl / duplicate) Protocol: melting curve ranging from 15°C to 100°C with increment of 0.5°C Estimated run time : 1h 35min Raw data Calculation of the negative first derivative Plate map Value of the Tm (inflection point of the melting transition)

Examples of application- to determine the Tm of a purified protein- to control the reproducibility of the protein production process (batch to batch)- to determine the impact of mutations on a protein folding- to screen buffer condition- to look at the effect of increasing concentration of urea on a protein

Tm of a purified protein Most proteins unfold between Negative control 40 – 90 °C. (buffer) Tm of a protein is characteristic for that protein in a determined Tm = 85°C condition of buffer. Reproducibility of this protein production process batch 1 Tm = 65°C Difference between two batch 2 Tm = 64.5°C Tm less than 1-2°C

Tm of Cys- mutated variants Protein containing Cys residues. Evaluation of Cys-mutated variants of this protein in order to look at the impact of the Cys residue in its stability. Negative control (buffer without protein) Protein Tm 52°C Mutant 1 (2 mutated Cys) Tm 49.5°C Mutant 2 (4 mutated Cys) Tm 45°C Mutant 3 (6 mutated Cys) aucun Tm The Tm decreases with the increase of the number of mutated Cysresidue indicating that the global conformation of the protein is affected by Cys mutation.

Screening of buffer conditions (1)Choice of a buffer condition for a protein Negative controls (buffers without protein) Protein in buffer A Tm 72°C Protein in buffer B Tm 71°C Protein in buffer C Tm 66°C Buffer C has bad effect on this protein, therefore it is avoided as buffer for this protein

Screening of buffer conditions (2)Tm of a protein with its substrate Negative controls (buffers without protein) Protein alone Tm 52°C Protein with its substrate Tm 35°C Protein with a non- hydrolyzable substrate analog Tm 52.5°C The presence of the substrate does not stabilize the protein.

Screening of buffer conditions (3)Choice of a preservative agent Negative controls (buffers without protein) Protein without preservative agent Tm 52°C Protein + perservative agent A Tm 29°C Protein + perservative agent B Tm 52.5°CBased on this results, preservative agent B is chosen for this protein.

Effect of urea conc. on a proteinWhich concentration of urea can partially disrupt the 3D structure ofthis protein ? [urea] Tm 0M 52°C 0.2 M 51°C 0.5 M 50°C 1M 48.5°C 2M 45.5°C 4M none 6M none [ urea] < 2 M disrupts partially the 3D structure of the protein.

ConclusionThis few examples of application with purified proteins show that wecan have quickly information about folding by using TSA.This tool is very helpful for determination of a Tm, buffer conditionscreening, determination of mutation impact on a protein … Advantages Drawbacks not time-consuming Because of the use of Sypro Orange, can not be used with easy to use hydrophobic compounds high throughput scale requires just a small device (like a CFX96)

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Understanding protein folding with thermal shift assays

by American Biotechnologist on Aug 18, 2011

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