This study investigated the structural changes that occur in the protein synaptotagmin-1 (syt1) when it binds lead instead of its native metal ion calcium. Researchers expressed a syt1-GST fusion protein in E. coli and purified it using affinity chromatography. Differential scanning calorimetry experiments showed syt1-GST unfolded in multiple stages both in the presence and absence of calcium, suggesting calcium does not stabilize the protein structure. Further experiments replacing calcium with lead are needed to understand how metal binding affects syt1 activity in neurotransmitter release.

1. Department of Chemistry and Biochemistry
Siena College, Loudonville, NY 12211
Investigation of the metal-induced structural change of Synaptotagmin-1
Alexandria Konkol, Chad Dashnaw, Kayla Provenzano, and Jesse W. Karr*
Abstract
Lead poisoning has been identified as one of the most common, yet
preventable health concerns in the U.S. Though lead has been known
to cause neurological problems, the mechanisms by which various
biological systems are affected by lead remains unknown; thus,
treatment for lead poisoning is inadequate. Various metalloproteins,
such as the membrane trafficking protein synaptotagmin I (syt1), have
been shown to bind lead with a greater affinity than their native metal
ion and have been identified as potential targets of lead. Normal
functioning syt1 will interact with another membrane trafficking
protein to facilitate the release of certain neurotransmitters; however,
in the presence of Pb(II), there appears to be a loss of interaction
between these two proteins. This research aims to better characterize
the structural change that occurs when syt1 binds lead rather than
calcium, the native metal ion and to study the reversibility of such
binding.
Neurotransmitter Release
adapted from Tucker et al. 2002
1) Formation of SNARE complex
• Synaptobrevin
• Syntaxin
• SNAP-25
2) Activation by calcium
3) Syt1 interpenetrates
membrane
4) Phospholipid bilayer is
deformed, allowing for a
“bulge” in the membrane
5) Syt1 binds SNARE complex,
further driving assimilation
6) Full endocytosis of the
neurotransmitter
• 421 amino acid long, 47 kDa
target membrane bound
protein
• Thought to be involved in
neurotransmitter release
• Known as to be a Ca(II) binding
protein
• Contains 2 C2 domains
• Believed to facilitate fusion
between neurotransmitter
vesicles and target membranes
• Shown to bind lead with higher
affinity than native ion
The quaternary structure of syt1. The cytoplasmic
region of syt1 (PDBid: 2R83) and its two calcium
binding domains, C2A (PDBid: 1BYN) and C2B
(PDBid: 1TJX).
C2A
C2B
Synaptotagmin-1 (syt1)
Protein Expression of Syt1-GST
Discussion and Conclusions
It appears that inducing E.Coli at a higher temperature provides a
greater yield of cells and thus, a greater protein yield. Although the
folded structure of syt1-GST induced at this temperature has yet to be
confirmed, we believe it actually better represent the biologically
active structure of syt1. Additionally, although the high salt wash is
able to remove the majority of the nucleic acid contaminate, we feel
the the purification process requires additional purification steps in
order to remove all mRNA contaminates.
Based on the calorimetric studies, it would appear that the presence
of Ca2+ does not stabilize the protein. To fully understand the activity
of syt1-GST in the presence of metal ions, the experiment should be
ran under the same conditions in the presence of Pb2+.
Acknowledgements
• Jonathon Pevsner of Johns Hopkins University for the plasmid
encoding the syt-GST (p65, 1-5) fusion protein.
• Siena Summer Scholars Program and CURCA for providing funding.
• Siena College Department of Chemistry and Biochemistry for use of
facilities.
• The SAInT Center at Siena College
References
• Chapman, E. R. (2008) How does synaptotagmin trigger neurotransmitter release? Annu. Rev. Biochem. 77,
615-641.
• Garcia, R. A., Forde, C. E., and Godwin, H. A. (2000) Calcium triggers an intramolecular association of the C2
domains in synaptotagmin. Proc. Nat. Acad. Sci. U.S.A. 97, 5883-5888.
• Bouton CM, Frelin LP, Forde CE, Arnold Godwin H, Pevsner J. (2001) Synaptotagmin I is a molecular target for
lead. J. Neurochem. 76, 1724-35.
• Sukumaran, S., Banerjee, s., Bhasker, S., Thekkuveettil, A. (2008) The cytoplasmic C2A domain of
synaptotagmin shows sequence specific interaction with its own mRNA. Biochem. Biophys. Res. Commun.
373, 509-514.
• Tucker, W. C., and Chapman, E. R. (2002) Role of synaptotagmin in Ca2+-triggered exocytosis. Biochem. J. 366,
1–13.
DSC Characterization of Syt1-GST
Amp-R Gene
Syt1-GST Gene
Insert Plasmid into
E. Coli Cell
Cells containing plasmid
express ampicillin
resistance
Grow cells on LBA agar plates
Only cells receiving plasmid
will form colonies on plate
• BL21 (DE3) and
RosettaTM(DE3)pLysS E. Coli
competent cells were transform
with syt1-GST encoding plasmid
• Transformed cells were initially
grown on a small scale:
• Miller’s Mix
• Optimal growth time: 10 hrs.
• Optimal growth temp: 37 °C
• Small scale growths were used to
for large scale growth and
expression:
• 2XYT Broth
• Induced with IPTG
• Optimal expression time: 4 hrs.
• Optimal expression temp: 37 °C
SDS-PAGE Analysis of Syt1-GST purification. A) Lanes 1-5: Recovered FPLC fractions from syt1-GST
induction at 37 °C; lane 6: molecular weight standards. B) Lanes 1-7: Recovered FPLC fractions from syt1-
GST induction at 30 °C; lane 8: BSA; lane 9 myoglobin; and lane 10: molecular weight standards.
Protein Purification of Syt1-GST
• Cells were lysed with a Sonic
Dismembrator
• Pulse: 1 sec. on, 1 sec. off pulse
sequence at 80% amplitude
• Duration: 1 min
• Clarified lysate was purified using a
GSTrap FF column
• Resin: Glutatione Sepharose Fast
Flow
• Wash Buffer: 2.8 mM Phosphate
buffer, pH 7 with 0.5 M NaCl, 2.7
mM KCl, and 5 mM EDTA
• Elution Buffer: 50 mM Tris buffer
pH 8 with 15 mM reduced
Glutathione
FPLC trace for purification of syt1-GST. The
280 nm signal is used to detect protein
concentration, the 254 nm signal is used to
detect DNA/RNA contamination.
Differential scanning calorimetry (DSC) experiments were used to
investigate the thermodynamic stability of syt1-GST in the presence
and absence of Ca2+.
DSC thermogram of 15 µM syt1-GST in
the presence of 90 mM CaCl2. This
thermogram suggests a two event
unfolding process which can be modeled
using TM values of 48 and 52 °C.
A
B
Schematic description for transformation of E. coli
competent cells with the syt1-GST plasmid.
DSC Thermogram of 15 µM syt1-GST in
the absence of any additional metal
ions. This thermogram suggests a three
event unfolding process which can be
modeled using TM values of 56, 66, and
70 °C.