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1www.thermofluor.org
Thermofluor (aka Differential Scanning Fluorimetry)
2www.thermofluor.org
Thermofluor
• Thermofluor gives an optical readout of protein melting as e.g. observedwith DSC.
• Optical readout is much more sensitive than direct thermal measurement.• TF consequently allows parallel measurements in 384 well-plates using
fluorescent imaging plate readers.
3www.thermofluor.org
ThermoFluor & DSC
TemperatureC
p (k
cal/m
ol/K
) Tm
Temperature
Fluorescence
Tm
DSC Measurement of Tm(Proteins melt cooperatively at
a Tm reflecting their stability)
4www.thermofluor.org
Thermofluor Applications
• Optimization of solution conditions (pH, Ionic strength, cofactors, metals, etc.) forprotein stabilization.
– Protein preparation and biochemistry– Optimization of crystallization conditions
• Measurement of ligand binding affinity– Drug screening– Analysis of binding cooperativity between multiple ligands
• Inhibitors plus substrates or cofactors.• Agonists vs. antagonists (e.g. NHR ligands vs. activator & repressor
peptides).– Use of “functional probe libraries” incorporating substrates, metabolic
intermediates, & cofactors to “decrypt” proteins of unknown function.– Use of structural analog libraries to determine detailed energetics of cofactor
binding (e.g. ATP bound to different kinases).
5www.thermofluor.org
Ligand-Induced Thermal Stabilization measuredusing Fluorescence Readout
heat
+ +
= Ligand = Dye
KA KDyeKU
hν
6www.thermofluor.org
Thermofluor Can Measure Protein Stabilization
hν
Native Protein
Protein stabilizedby environment
Melted Protein (Dye Bound)
Energy
Folded Melted
Stabilization due modification of environment
7www.thermofluor.org
Ligand Binding Can Stabilize Protein Structure
Ligand
Temperature
Fluorescence
Tm
Tm of Protein:Drug
Complex
ΔTm
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Thermofluor Can Measure Energetics of Ligand Binding
hν
Native Protein
Protein w/Ligand Bound
Melted Protein (Dye Bound)
Energy
Folded Melted
Stabilization due to Ligand Binding
9www.thermofluor.org
Thermofluor Applications
ΔTm
pH[I]
Protein Stability Optimization
100uM 50uM 25uM NoNo
1uM10uM
100uM
01
2
3
4
5
6
7
8
9
10
[Inhibitor] [Sub
stra
te 1]
Ave
rag
e A
vera
ge ΔΔ
Tm
Tm
Analysis of cooperativity between substrate and inhibitor binding
ΔTm
Drug [email protected]
10www.thermofluor.org
Multiple Ligand Binding
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Differentiating “Activators” & “Repressors”(e.g. for Nuclear Hormone Receptors)
12www.thermofluor.org
Mapping Kinase ATP Binding Energetics by Thermofluor using an ATP Analog Library
adenosine
ADPATP
2’deoxyadenosine
5-aminoimidazole-4-carboxamide-1-B-4-ribofuranoside
deoxyguanosine xanthinehypoxanthine
adenosine
ADPATP
2’deoxyadenosine
5-aminoimidazole-4-carboxamide-1-B-4-ribofuranoside
dATP, dADP
ATPγSAMP-PNP
Kinase “A” Kinase “B”Conclusion: Different kinases have evolved different specific interactions to maintain ATP-binding capacity.Implication: ATP-analog drugs can be specific for individual kinases. (Thought for many years to be untrue.)
13www.thermofluor.org
Thermofluor-Assisted High-Throughput Crystallography
Protein
Stability Screen(pH, [I], EDTA, etc.)
Functional Probe Library
(Cofactors, Metabolites)
TF Heavy Atom Screen
DataBase Querye.g. Riken Heavy Atom DB
Crystals
SAD, SIRASPhase Determination
3D Structure
14www.thermofluor.org
References
• High Density Miniaturized Thermal Shift Assay as a General Strategy for DrugDiscovery, M.P. Pantoliano, E. Petrella, J. Kwasnoski, V. Lobannov, J. Myslik, E. Graf, T.Carver, E. Asel, B. Springer, F.R. Salemme Journal of Biomolecular Screening. 2001;6(6):492-440
• Direct Binding Assays for Pharma Screening. M.J. Todd & F.R. Salemme, GeneticEngineering News. 2003;3:28-29
• Thermodynamic Stability Of Carbonic Anhydrase: Measurements Of Binding AffinityAnd Stoichiometry Using Thermofluor, Matulis D, Kranz JK, Salemme FR, & Todd MJBiochemistry 2005; 44(13): 5258-66.
• Decrypting the Biochemical Function of an Essential Gene from Streptococcuspneumoniae using Thermofluor Technology, Carver TE, Bordeau B, Cummings MD,Petrella EC, Pucci MJ, Zawadzke LE, Dougherty BA, Tredup JA, Bryson JW, Yanchunas JJr, Doyle ML, Witmer MR, Nelen MI, Desjarlais RL, Jaeger EP, Devine H, Asel ED, SpringerBA, Bone R, Salemme FR, Todd M. J Biol Chem. 2005; 280:11704-11712
• Applications of Calorimetric Methods to Drug Discovery and the Study of ProteinInteractions. P.C. Weber & F.R. Salemme, Curr. Opinion in Structural Biol. 2003; 13:115-121
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