Glyphosate research papers - Compiled by Dr.Alex Vasquez and Dr Eva Sirinaths...
Sterics Poster
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Introduction and Methods
Introduction
Many new biomolecules are created by conjugating proteins to
synthetic polymers. One important method of biomolecule
conjugation is disulfide exchange between a thiol on a biomolecule
and an activated disulfide present on a polymer. From previous
studies in our lab, when these disulfide polymers were included in
the network of a hydrogel, incorporation of proteins by disulfide
exchange was sterically hindered. We were therefore interested in
the relative steric effect of the different components in disulfide
exchange. We investigated the differences in both the exchange rate
and maximum extent of disulfide exchange depending on three
variables: pH, thiol size, and polymer size.
Steric Effects in Peptide and Protein Exchange with Activated Disulfides
Jason Kerr, Jessica L. Schlosser, Donald R. Griffin, Darice Y. Wong and Andrea M. Kasko
Department of Bioengineering , University of California Los Angeles
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Kinetic Rate Constants
Figure 1: Kinetic rate constants for each variable
averaged over all other factors.
Figure 2: Kinetic rate constants for individual experimental conditions, grouped by thiol.
Figure 3: Maximum extent of exchange for each variable
averaged over all other factors.
Figure 4: Maximum extent of exchange for individual experimental conditions, grouped by thiol. Figure 5: Illustrations of protein-polymer pairings representative of exchange behavior
Maximum Disulfide Exchange Overall Comparison
Scheme 1: Chemical synthesis of PDG-SS-Pyr with different macromer sizes for comparison of the effect of PEG chain
lengths on disulfide exchange kinetics.
Synthesis and Exchange Conditions
pH and Polymer have mild influence on exchange
Protein S-Protein SH
Table 1: Relative influence of each factor on the kinetic rate constants and maximum extent of disulfide exchange.
(✓= influence, ✗= no influence)
The relative influence of 3 factors on the kinetics of disulfide exchange were
investigated: 1) thiol size and steric accessibility, 2) macromer size, and 3)
pH. Kinetic rate constants increase with increasing pH, but maximum
exchange is not affected by pH in the range tested. Small molecule disulfides
react faster and more completely than disulfides with polymer chains, but
increasing sizes of the chains has no effect. Larger proteins, and particularly
proteins with additional local steric barriers react more slowly and
incompletely. With decreasing pH and increasing protein size, the disulfide
size becomes less important in determining the kinetics of disulfide exchange.
Overall, the sterics of the proteins, at any given pH, are more influential than
macromer size and pH in determining the rate and extent of disulfide
exchange.
Factors
Influence on Kinetics Influence on Max
Exchange
pH ✓✓ ✗
Protein ✓✓✓ ✓✓✓
Macromer ✓ ✗
Thiol access dominates exchange
S-S
S-S
= pyridine
= o-NB
SH
SH
SUCC-SS-Pyr
PEG-PDG-SS-Pyr
GSH
βLG
+
+
kexch ~ 0.035 s-1
kexch ~ 0.010 s-1
max exch. ~100%
fastest, most complete
sterics negligible
max exch. ~ 75%
moderate, variable
thiol and disulfide sterics
interact
S-S
PEG-PDG-SS-Pyr
+ SH
BSA
kexch ~ 0.001 s-1
max exch. ~ 30%
slowest, least complete
thiol sterics dominate
pH = 8.0
pH = 8.0
all pH
This material is based upon work supported by: the National
Institutes of Health (NIH) and the Milton Gottlieb Scholarship
Fund.
! pH = ! k
! Thiol access = ! k
! PEG length ≠ ! k
! pH ≠ ! max exchange
! Thiol access = ! max exchange
! PEG length ≠ ! max exchange
! pH = ! k but ! pH ≠ ! max exchange
Higher pH deprotonates the thiol, increasing the ratio of
thiolate anion, thus rate of disulfide exchange, but not
maximum disulfide exchange.
! PEG length ≠ ! k and ! PEG length ≠ ! max exchange
PEG chain has a moderate affect on k and max exchange vs.
no PEG chain, but their flexibility and distance from the
disulfide bond means differences in chain length are
relatively unimportant.
! Thiol access = ! k and ! max exchange
Local and global steric conditions affect thiol accessibility. SN2 reactions depend on access and
positioning. This factor is most influential.
PEGProtein S
S S
S Protein
Results
Conclusion Acknowledgements
O
O
O
O
OH
NO2
O
O
O
O
O
NO2
EDC/DMAP
DCM
24hr, RT O
O
O
O
O
O2N
n
HO
O
O
O
O
NO2
O
O
O
O
O
O2N
OH
n
NaBH4
N S
S
O
O
O
OH
O
O
O
O
O
NO2
O
O
O
O
O
O2N
O
n
N S
S
O
O
O
NS
S
O
O
O
DCM
24hr, RT
EDC/DMAP
DCM
24hr, RT
O
n= 45, 91, 227
Compound A Compound B
O
HO OH
n
O
O
O
O
O
NO2
O
O
O
O
O
O2N
O
n
N S
S
O
O
O
NS
S
O
O
O
n= 44, 90, 226
N S
S
O
O
O
OH
R-SH
pH
6.0
7.0
7.4
8.0
O
O
O
O
O
NO2
O
O
O
O
O
O2N
O
n
R
S
S
O
O
O
R
S
S
O
O
O
n= 44, 90, 226
R
S
S
O
O
O
OH
NH
S
Monitored at 340nm
or
or
R=GSH, βLG ,BSA
Scheme 2: Experimental disulfide exchange conditions. Reaction rate constants
were determined from change in absorbance of pyridine-2-thione measured at
340nm when released from the macromers.
!
References
[1] PDB ID: 2Q2M, Vijayalakshmi, L., Krisha, R., Sankarayarayananan, R., Vijayan, M.
Proteins 71: 214-249 (2007).
[2] PDB ID: 3NPO, Loch, J., Lewinski, K. J. Mol Recognit. 24: 341-349 (2011).
[3] PDB ID: 4F5S, Bujacz, A. Acta Crystalogr. Sect. D. 68: 1278-1289 (2012).
[4] PDB ID: 3V03, Majorek. K.A., Porebski, P.J., Chruszcs, M., Almo, S.C., Minor, W. Mol.
Immunol. 52: 174-182 (2012).
Glutathione
~50%!dimer!!!!!!!!!!!!!!!!!!!!!!~50%!monomer!
Bovine Serum Albuminβ-Lactoglobulin
100%!dimer!!!!!!!!!!!!!!!!!!!!!!100%!monomer!
DTT!
[1]! [2]! [3]! [4]!
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