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Gary J. PielakUNC-Chapel Hill
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• Crowding theories old & new• Weak (quinary) interactions• 19F NMR & drkN SH3• Stability in cells & in vitro
under crowded conditions
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• Chemical (quinary) surface interactions are fundamental to stability
in cells & under crowded conditions in vitro
• Gram for gram, monomers more stabilizing than polymers
• Common cosolutes ≠ physiological information
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• Cellular chemistry occurs in cytoplasmo 300 gL-1 macromolecules
• Biochemistryo < 10 gL-1
• Cytoplasm could change :o Structureo Stability McGuffee & Elcock, PLoS Comp Biol 2010
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synthetic polymers globular proteins cytoplasm
PVP:
Ficoll: highly-branched sucroseDextranPEG
lysozymeBSAovalbumin
cellslysate
ControlsSugars, urea, ethylene glycol, trimethylamine oxide (TMAO)
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Oil drop model• Interior hydrophobic &
well packed
• Exterior• “just” hydrophilic
Stability: N UKu = [U]/[N]; ΔGo’u = -RTln(Ku)
U
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Quaternary
NH3+
COO-Ala Ser Lys Tyr Gly
PrimaryTertiary
Secondary
McGuffee, Elcock, Plos Comput Biol 2012PDBID 1GB1 & 1Q10
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9
Sucrose → Ficoll
Volume excluded to test molecule
COMPACTIONcrowder (rtest + rcrowder)3
α excluded volume
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“native conformation is determined by the totality of interatomic interactions…in a given environment” protein can only be properly understood “under conditions similar to those for which it was selected – the so-called physiological state”
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(1980) Biophys J 32: 347-360.
“The next level, quinary structure, would relate to the interactions in helical fibers, as occur in the sickle cell form of hemoglobin.
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Sharp KA (2016)Unpacking the origins of in-cell crowding.
PNAS 113:1684-1685.
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0
00
• Repulsive• charge-charge
• Attractive• charge-charge• H-bonds,• hydrophobic
+
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Quaternary
NH3+
COO-Ala Ser Lys Tyr Gly
PrimaryTertiary
Secondary
Quinary
McGuffee, Elcock, Plos Comput Biol 2012PDBID 1GB1 & 1Q10
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Uncertainties
TriplicateStandard deviations of the meanMonte Carlo analysis
16ΔGDo’ at residue level without heat by using ...
Usually accomplished with heat or denaturants, but under crowded conditions ...
Ku = [D]/[N]Stability: ΔGo’u = -RTln(Ku)
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Rachel Cohen& …
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• Label with 19Fo 5-fluoroindole 5F-trp
• Area ~ [species]
Ku
124 125 126 ppm
Monteith et al. Chem. Comm. 2012
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• 59 amino acids, 6.8 kDa, pI 5• NMR structure known• Dynamic ensemble in unfolded state• ΔGu°’ ≈ 0 kcal/mol
Evanics et al. Biochemistry 2006Crowhurst JMB 2002
Solvent exposure of trp changes
Folded Unfolded
Austin Smith
Annelise Gorensek
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Buffer
Urea
TMAO
pH 7.2
100 g/L
Only 3 hours,reversible
50 g/L
Smith et al., PNAS, 2016
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buffer
cells uncorrected
300 g/L glucose/dextran300 g/L sucrose/Ficoll200 g/L ethylene glycol /PEG
Smith et al., PNAS, 2016
22Smith et al., PNAS, 2016
Stabilizing Destabilizing Can’t tell
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• Gram for gram, monomers ≥ polymers• Enthalpic component• No pattern with respect to entropy
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resuspendinduce expression harvest
cell pelletLabeled/enriched protein
in-cell NMR spectrum
pellet sample &
check supernatant for leakage
M9 +19F, 15N, etc.
19F spectrum
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280 290 300 310
100 g/L
ΔGo’
Dkc
al/m
ol
Temperature K
Reality in cells between corrected &raw
Smith et al., PNAS, 2016
As, or less, stable in cellsNot as expected from simple theory5o important in cellsNot like synthetic polymers
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SH3, pI 5
100 g/LBSA, pI 6Lysozyme, pI 9
Smith et al., PNAS, 2016
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Stability of CI2In 100 g/L solutions of
Weak interaction & Dynamics of Calmodulin
0
00
Latham, Kay JBNMR 2013
ΔΔG
uo’
kcal
/mol
Sarkar, Smith & Pielak PNAS 2013Sarkar, Lu & Pielak Biochemistry 2014
Monteith et al. 2014
GB1
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• Chemical (quinary) surface interactions are fundamental to stability
in cells & under crowded & confined conditions in vitro
• Gram for gram, monomers more stabilizing than polymers
• Common cosolutes ≠ physiological information
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• Drs. Greg Young &• Marc ter Horst