gary j. pielak unc-chapel hill · mcguffee, elcock, plos comput biol 2012. pdbid 1gb1 & 1q10....

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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