tracking protein allostery in evolution glycogen phosphorylase frees sugars to provide energy gp...
TRANSCRIPT
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Tracking Protein Allostery in Evolution
• Glycogen phosphorylase frees sugars to provide energy
• GP orthologs diverged 600,000,000 years can respond to transcription controls, metabolite concentrations and post translational modifications
Metabolites are Glucose, AMP, ATP and PTMS are Ser or Thr Phosphorylation
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Antique Phosphorylase
• non allosteric; transcriptional control
• binding site for maltodextrose magenta
• PLP cofactor- vitamin B6, (yellow) - side chain of Lys-680
• The active site between the N-terminal and C-terminal domains
• maltose -purple
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Yeast GP has controls
Phospho Thr N-terminal residues 1-22, corresponding to the N-terminus of muscle phosphorylase, ribbon in white. The unique N-terminal extension of yeast phosphorylase (residues -1 through -39) is drawn as a ribbon in pink.
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Yeast GP has controls
Phospho Thr
Unphosphorylated enzyme: N-terminal extension blocks the active site. Phosphorylation moves the N-terminal extension to subunit interface to displace the inhibitor, glucose 6-phosphate (orange).
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Regulated GPs have two states, inactive and active
How is the switch between conformations made?
Triggers can vary, the response must settle the chemistry of catalysis
Yeast and human GPs use the same mechanism to stabilize the active conformation- a cluster of hydrophobic side chains form, but from different starting configurations
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Orange hydrophobic residues cluster in
active Yeast GP
Clustered Distributed
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Given the built-in conformational change
It is easy to engineer a new allosteric trigger
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Engineering an allosteric switch
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Ser Pi can activate, AMP can activate, in mutant only Ni ion activates
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A baroque assembly of chaperone and client
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SptP binds unfolded to 3 SicP chaperones
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How is a universal protease inhibitor made?
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Multiple interfaces of ecotin
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Ecotin- domain swapped tetramer
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A chain is as strong as its weakest linkA net is stronger than its strongest link
• Chain • Tetramer net
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A single side chain can configure a functional protein interface
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Ecotin bound thrombin molecule red – Moves 60’s loop
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Analytical Ultracentrifuge and Gel chromatography are simplest ways to
measure Mr in solution
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AUC and GEL Chromatography
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Caffeine Mw 194Binds to 100,000protein
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SPR measures binding of small molecules to proteinSensitivity 100 D relative to 100,000 D
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Technology Behind Biacore
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Chip structure
Glass
Linker layer
Gold
Dextran layer
Specific layer
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SPR Detection System
1 RU ~ 1 pg/mm2
1 RU = 0.0001º
Reflected Light
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Information in a Sensorgram
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Measuring added mass by lightThe Octet
• BioLayer Interferometry (BLI) monitors the binding of proteins and other biomolecules to their partners directly in real time.
• binding interaction continuously monitored by measuring the change in thickness of the protein layer on the biosensor tip
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The Octet has Crude Sample Compatibility
• Only molecules that bind to or dissociate from the biosensor surface produce a signal change.
• Proteins can be assayed in crude mixtures (cell lysates) or in DMSO (up to 10%), and glycerol, reducing sample preparation
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Octet Biosensor measures Ligand induced protein to protein disociation
surface
peptide
protein
compoundConcept: inhibitor compound changes protein structure to release protein from binding partner
10 M compound
5 M compound
0 M compound
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Free energy calculations for the three complexes
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Free Energy For Proteins Interacting
Horton And Lewis
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Two States in Association
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Fits of Buried Surface to Experimental Free Energy
hydrophobic atoms alone correlate poorly with the free energy of association
Counting the polar and nonpolar buried surface, and the weighting factors, the correlation is 96%.
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Conclusions
• buried polar atoms contribute favorably if paired, unfavorably in unpaired and hydrophobic atoms contribute favorably.
• entropy lost on complex formation is not well explained- it was made an adjustable parameter in these studies: one of a total of 3 parameters for 15 observable.
• H bond average is -0.24 kcal/mole. Range is 0 to -.71: 44 of the bonds are charged and contribute -0.8 to -1.5.
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Structural and thermodynamic characterization of
free and complexed ACTR and CBP
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Solution structure of the ACTR–CBP complex- ACTR is pink and CBP blue
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Sequences Compared
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Interface interactions