Probing Single-Molecule Protein Spontaneous Folding-Unfolding
Conformational Fluctuation Dynamics: The Multiple-State and
Multiple-Pathway Energy Landscape
Zijian Wang, H. Peter Lu*
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green,
Ohio 43403
Supporting information
S1. Ensemble titration curve measurements.
We carries out the ensemble titration curve measurement of CaM in solution on an
inverted confocal microscope (Axiovert 200, Zeiss) that uses a laser (532nm CW) as the light
source for excitation. The Laser beam focuses through a 100× oil immersion objective lens (1.3
NA, 100×, Zeiss) onto the upper surface of cover slip after the excitation light is reflected up by
a dichroic beam splitter (z532rdc, Chroma Technology). Solution is prepared with different
concentration of denaturant solvent GdmCl in a sample chamber using the above set-up to
measure ensemble EFRET.
We take ensemble-average measurements in solution by keep
recording minute time scale D-A fluorescence intensity trajectories mixed with different
concentration of denaturant GdmCl. By building a histogram of calculated EFRET trajectories
from two-band D-A fluorescence intensity trajectories, we obtain an ensemble-averaged EFRET
value at different concentration of GdmCl. Figure S1 shows our ensemble-averaged measured
EFRET as a function of denaturant concentration. We see 2M GdmCl as the titration midpoint of
equally populated folded and unfolded CaM molecules. We further use 2M GdmCl condition as
our
experimental
condition
for
probing
equilibrium
spontaneous
folding-unfolding
Mean FRET Efficiency
conformational fluctuations of single-molecule CaM embedded in agarose gel.
0.7
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0.5
0.4
0.3
0.2
0.1
0
1
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5
6
Concentration of GdmCl (M)
Figure S1. Ensemble titration curve in solution incubated with different concentration of
denaturant solvent GdmCl. The EFRET is plotted with GdmCl concentration. We reach the
titration midpoint at 2M of GdmCl.
S2. Sample preparation for single-molecule imaging
The samples for single-molecule conformational folding-unfolding dynamics measurements
were prepared inside a 1% agarose gel with 99% of buffer solution (Type VII, Sigma). We made
samples of CaM into different concentrations of denaturant GdmCl in the mixture of 1 nM CaM,
1.25µL and oxygen scavenger with Trolox solution to obtain a 10µL mixture of protein and
denaturant solvent. The Trolox solution was prepared previously by dissolving about 1 mM 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) in 1 mg/mL glucose oxidase,
0.8% D-glucose and 0.04 mg/mL catalase in order to protect fluorescent dye from
photobleaching or blinking as a result of triplet state oxygen quenching as well as other
photophysical processes. We then heated the 10µL 1% agarose gel just above its gel-transition
temperature (26°C) and quickly mix the above protein solution with denaturant solution and the
gel between two clean cover glasses to form a sandwiched sample. All solutions were prepared
with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) at pH 7.4 with 1mM
EGTA. To probe conformational dynamics of single-molecule protein at different concentration
of denaturant solvent, we carried out concentration dependent experiments with different ratio of
mixture in the sample which is listed below.
～1 nM CaM+0M
Gdmcl
～1 nM CaM+1M
Gdmcl
～1 nM CaM+2M
Gdmcl
CaM
GdmCl
Trolox+Oxygen
Scavenger
Agarose
Gel
0.2µL 200nM
0µL 8M
9.8µL
10µL
0.2µL 200nM
2.5µL 8M
7.3µL
10µL
0.2µL 200nM
5µL 8M
4.8µL
10µL