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Proteins Structures under Electrospray Conditions

Alexandra Patriksson, Erik Marklund, Carl Caleman & David van der Spoel [email protected]

Protein Dehydration

?

Principle of Electrospray

Diagrams and text (partial) by Dr Paul Gates, School of Chemistry, University of Bristol

Rayleigh Limit

q 2 = 64 2 r3

· yields a maximum of 4-5 charges for a cluster of 216 molecules in vacuum

4 Ammonium in Water

6 Phosphate in Water

Why Study Electrospray?

· Not a lot is known about the conformations of proteins in vacuo · Electrospray will be used as the sample injection device for X-FEL · Difficult to study experimentally, hence Molecular Dynamics (MD) simulations are a useful tool

Bio-Imaging with X-FEL

X-FEL

· Extremely high intensity (10 orders of magnitude larger than synchrotron) · Sample will be destroyed due to radiation damage · Take diffraction image in femtoseconds before sample explodes (Neutze et al. Nature 406, p. 752 (2000))

X-FEL Proof of Principle

30 nm radiation on artificial sample

First shot

Second shot

Reconstruction

Sample (EM) Reconstructed image Chapman et al. Nature Phys. 2, p. 839 (2006)

Proteins Studied

· Lysozyme · Ubiquitin · Insulin · C-terminal fragment of L7/L12 ribosomal protein (CTF) · Trp-cage

Protein Details

PDB entry Protein #AA Resolution Source Hen egg white Homo sapiens E. Coli 1AKI Lysozyme 129 1.50Å

1UBQ

Ubiquitin Ribosomal L7/L12 Insulin - AB chain Trp-cage

79

1.80Å

1CTF

74

1.70Å

4INS

51

1.50Å

Pig Synthetic construct

1L2Y

20

NMR

Simulations

· OPLS/AA force field + TIP4P water · PME in bulk simulations · No cut-off in vacuo · All vacuum simulations performed 3 times with different starting coordinates · GROMACS software

Simulations

· Bulk solution - with PBC · 6 Å water shell - in vacuo · 3 Å water shell - in vacuo · Dry - 2 sets of 3 simulations

Charges in Vacuo

· Two different charge sets were used in vacuo for the dry protein: · Solution charges · Vacuum charges

Charges in Vacuo

· Limited experimental data for charges on Lysozyme and Ubiquitin, not for the other proteins · Computer generated charges based on electrostatic potential and gas-phase basicity

Determining Charge Sites

· For N titratable groups with M charges there are N!/[(N-M)! M!] possibilities · The actual charge state is determined by the Boltzmann weight · In addition to Lys and Arg also His and Gln may be protonated

Net Charges

Protein Lysozyme Ubiquitin CTF Insulin Trp-cage Solution +8 0 -2 -2 +1 Vacuum +8 +7 +5 +5 +2

Lysozyme

Solution charges Vacuum charges

Ubiquitin

Solution charges Vacuum charges

CTF

Solution charges Vacuum charges

Insulin

Solution charges Vacuum charges

Trp-cage

Solution charges Vacuum charges

Results

· Evaporation & Cooling · Root mean square deviation · Secondary structure · Vacuum structures

Evaporated - 10 ns

6 Å shell 3 Å shell Lysozyme 114 / 1042 (11 %) 55 / 317 (17 %)

Ubiquitin

68 / 770 (9 %)

37 / 254 (15 %)

CTF

69 / 681 (10 %)

31 / 217 (14 %)

Insulin

62 / 586 (11 %)

31 / 161 (19 %)

Trp-Cage

39 / 342 (11 %)

21 / 95 (22 %)

Final Temperature

6 Å shell 3 Å shell Lysozyme 266 (3) K 264 (4) K

Ubiquitin

267 (4) K

264 (4) K

CTF

263 (4) K

262 (5) K

Insulin

265 (4) K

261 (6) K

Trp-Cage

258 (4) K

243 (8) K

Root mean square deviation of Ca atoms in bulk water

Average RMSD (Å)

Bulk 6 Å shell 3 Å shell 0 Å qsol 0 Å qvac Lysozyme 1.3 1.4 1.5 3.0 N/A

Ubiquitin

1.7

2.2

2.2

2.5

4.6

CTF

1.6

1.5

1.8

4.1

3.7

Insulin

4.0

4.4

4.7

5.0

5.1

Trp-Cage

2.8

2.4

2.5

4.1

3.7

Secondary Structure

(qvac)

Comparison of charge sets

Vacuum Structures

Lysozyme CTF Trp-cage

Ubiquitin

Insulin

Evaporation from Ubiquitin

Bio-Imaging with X-FEL

Resolvating Dehydrated Proteins in Silico

· Use vacuum structures obtained from reconstruction algorithms · Resolvate in the computer [1] · Find Gibbs energy minimum [2]

1. Y. Mao, M. A. Ratner and M.F. Jarrold: Molecular Dynamics Simulations of the Rehydration of Folded and Unfolded Cytochrome c Ions in the Vapor Phase J. Am. Chem. Soc. 123, 6503-6507 (2001) 2. M. Seibert, A. Patriksson, B. Hess and D. van der Spoel, Reproducible polypeptide folding and structure prediction using molecular dynamics simulations J. Mol. Biol. 354 pp. 173-183 (2005)

Resolvating Dehydrated Trp-cage

Native Simulated

Conclusions

· Significant rearrangement of sidechains and loop regions leading to 3-5Å RMSD · Main chain hydrogen bonds maintained in vacuum · A small water shell (3Å) is sufficient to maintain solution structure to a large degree · Computational resolvation feasible

A. Patriksson, E. Marklund & D. van der Spoel, Biochemistry 46, p. 933 (2007)

That's all folks

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