1

Gary J. PielakUNC-Chapel Hill

2

• Crowding theories old & new• Weak (quinary) interactions• 19F NMR & drkN SH3• Stability in cells & in vitro

under crowded conditions

3

• 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

4

• 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

5

synthetic polymers globular proteins cytoplasm

PVP:

Ficoll: highly-branched sucroseDextranPEG

lysozymeBSAovalbumin

cellslysate

ControlsSugars, urea, ethylene glycol, trimethylamine oxide (TMAO)

6

Oil drop model• Interior hydrophobic &

well packed

• Exterior• “just” hydrophilic

Stability: N UKu = [U]/[N]; ΔGo’u = -RTln(Ku)

U

7

Quaternary

NH3+

COO-Ala Ser Lys Tyr Gly

PrimaryTertiary

Secondary

McGuffee, Elcock, Plos Comput Biol 2012PDBID 1GB1 & 1Q10

8

9

Sucrose → Ficoll

Volume excluded to test molecule

COMPACTIONcrowder (rtest + rcrowder)3

α excluded volume

10

“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.

12

Sharp KA (2016)Unpacking the origins of in-cell crowding.

PNAS 113:1684-1685.

13

0

00

• Repulsive• charge-charge

• Attractive• charge-charge• H-bonds,• hydrophobic

+

14

Quaternary

NH3+

COO-Ala Ser Lys Tyr Gly

PrimaryTertiary

Secondary

Quinary

McGuffee, Elcock, Plos Comput Biol 2012PDBID 1GB1 & 1Q10

15

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

21

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

27

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

29

• Drs. Greg Young &• Marc ter Horst