Preliminary Experiments:

1
IDENTIFYING THE ROLE OF CALCIUM DURING THE
REDUCTION OF THE hN1LNRA PROTEIN Angie Seo,
Wellesley College Advisor Dr. Didem Vardar-Ulu
Results
Abstract Notch receptors are transmembrane
glycoproteins. Lin12/Notch Repeats are three
unique protein modules that help to regulate the
ligand induced proteolytic cleavage of the Notch
receptor.1 hN1LNRA is the first of the three
tandem Lin12/Notch Repeats in the human Notch1
receptor that regulates cell fate decisions.
Previous works have shown that Ca2 is required
for the correct folding of this repeat via a
unique set of three disulfide bonds.2 The goal of
this project is to investigate the role of Ca2
in stabilizing these disulfide bonds in the
folded protein. Our hypothesis is that the amount
of Ca2 in the environment will impact the
minimum redox potential and/or the time required
to reduce hN1LNRA. To test this hypothesis we
have exposed folded hN1LNRA in varying amounts of
Ca2 to identical predetermined reducing
conditions under anaerobic conditions. We assayed
samples at various time points using High
Performance Liquid Chromatography (HPLC) and
quantified the ratio of folded vs. reduced
protein to determine how the concentration of
Ca2 in the environment affected this ratio.
Time Time Time Time Time Time Time
0 min. 15 min. 30 min. 45 min. 1 hr. 2hr. 3 hr.
10然 21.4 LNRA-Ca2 complex folded 98.49 96.26 100 3.25 0 0 0
10然 21.4 LNRA-Ca2 complex reduced 0 0 0 96.75 100 99.41 98.41
10然 21.4 LNRA-Ca2 complex misfolded 1.51 3.74 0 0 0 0.59 1.58
50然 70.0 LNRA-Ca2 complex folded 99.49 69.38 42.14 23.46 17.26 4.12 0.80
50然 70.0 LNRA-Ca2 complex reduced 0 29.77 57.86 76.54 81.57 95.33 96.94
50然 70.0 LNRA-Ca2 complex misfolded 0.51 0.85 0 0 1.17 0.55 2.26
100然 77.9 LNRA-Ca2 complex folded - - - - 10.13 0.72 1.09
100然 77.9 LNRA-Ca2 complex reduced - - - - 89.87 98.62 98.91
100然 77.9 LNRA-Ca2 complex misfolded - - - - 0 0.66 0
500然 95.1 LNRA-Ca2 complex folded 98.94 98.90 79.88 69.08 62.39 36.79 24.77
500然 95.1 LNRA-Ca2 complex reduced 0 0 18.96 29.55 36.79 63.21 75.23
500然 95.1 LNRA-Ca2 complex misfolded 1.06 1.1 1.16 1.37 0.82 0 0
1 mM 97.5 LNRA-Ca2 complex folded - - - - 67.76 44.45 30.85
1 mM 97.5 LNRA-Ca2 complex reduced - - - - 29.16 52.94 67.80
1 mM 97.5 LNRA-Ca2 complex misfolded - - - - 3.08 2.61 1.35
Overlay of Chromatograms for Reduction of LNRA at
Redox Potential of -320