Supplemental Figures, Legends and Supporting Data · Supplemental Figure 1B. Effect of imidazole...
Transcript of Supplemental Figures, Legends and Supporting Data · Supplemental Figure 1B. Effect of imidazole...
Supplemental Figure Legends and Supporting Data.
Verification of QD-MBP-5HIS complex formation and protein:QD ratio. MBP95C-
Cy3 was utilized to monitor of MBP concentration via an absorbance maximum that
would not be masked by QD absorption at ~280 nm. One hundred pmol of 560 QD were
mixed with the molar equivalent amount of MBP95C-Cy3 sufficient to coat each QD
with ~10 MBPs. After mixing, the 560 QD–10MPBCy3 complex was allowed to self
assemble. A control was set up at the same concentration of MBP95C-Cy3, but without
the QDs. The measured absorbance of each solution was equal at the Cy3 absorbance
maximum ~ 553 nm. Each solution was added to an Amicon Centricon spin dialysis unit
(100 kD MWCO) and centrifuged for 20 min at 1000 x g. A wash of borate buffer was
added, followed by a second spin. The dialysate passing through the membrane was
collected and the amount of MBP95C-Cy3 in each sample was determined. More than
95% of the MBP95C-Cy3 in the non-QD control sample passed through the membrane.
In the 560 QD-10 MBP95C-Cy3 sample, less than 3% of the MBP95C-Cy3 present
passed through the membrane. Since the MW of MBP95C-Cy3 is ~ 45 kD and the MW
of the 560 QD-10 MBP95C-Cy3 is estimated to be >500 kD, these results support the
conclusion, together with the data on PL enhancement (Figure 1), that virtually all the
MBP95C-Cy3 present in the sample with the QDs was bound to the QD’s and therefore
could not pass through the membrane. Furthermore, the ratio of MBP95C-Cy3:QD is
~10, since if fewer than 10 MBP95C-Cy3s bound per QD, then a larger percentage of the
MBP95C-Cy3 present in the sample would be expected to pass through the membrane.
For example, if only 9 out of the 10 MBP95C-Cy3s adhered to the QDs, then 10% of the
protein in the experiment would pass through the membrane. It is important to recognize
that on average there are ~10 MBPs around each QD, and that some complexes might
have only 8 to 9 MBPs while others may have 11-12. A Gaussian distribution centered
around ~10 MPBs/QD is a plausible working model.
Supplemental Figure 1B. Effect of imidazole concentration on QD-MBP conjugate formation (fluorescence measured after amylose column)
Wavelength nm (400 nm excitation)450 500 550 600 650
PL
(AU
)
0
1x106
2x106
3x106
4x106
5x106
A.
450 500 550 600 650
PL (A
U)
0
10x106
20x106
30x106
40x106
50x106
MBP-5HIS Ratio of MBP to QD -->
MBP<-- Ratio of MBP to QD
604020
60
PL (A
U)
10x107
40
20
105
1
2
50 mM imidazole
20 mM imidazole10 mM imidazole5 mM imidazole
560 QD + 20 MBP/QD (Post amylose column)
560 QD w/ 20 MBP/QD(Before amylose column)
1 mM imidazole 560 QD w/ 20 MBP/Dot (Washed with 250 mM imidazole while on amylose column)
Supplemental Figure 1A. Comparison of the ability of MBP-5 HIS (penta-histidine) to adhere to QDs compared to MBP (penta-histidine truncation) (fluorescence measured after amylose column)
0
Wavelength nm (400 nm excitation)
20x107
30x107
40x107
50x107
Supplemental Figure 1A. Comparison of MBP-5HIS (penta-histidine) and MBP (penta-
histidine truncated) binding to DHLA-QDs. After the indicated ratios of each protein
were allowed to bind to 560 QDs, the QD-protein complexes were applied to an cross-
linked amylose resin column, washed with buffer and eluted with buffer + 10 mM
maltose. The amount of MBP-QD conjugate specifically eluted was determined by
measuring the PL of the eluted material. QDs without an MBP coating become
irreversibly entrapped in the matrix and will not pass through the amylose column with or
without application of maltose. Note: different x-axis scales are utilized to show the
large difference in concentrations eluted; the scale for MBP-5HIS-QD PL is 10-fold
expanded. (1B) Effect of imidazole concentration on QD-MBP coordination. MBP-
5HIS were allowed to coat 560 QDs at a ratio of 20 proteins per QD in the presence of
the increasing amounts of imidazole as indicated. These conjugated QDs were then
passed over amylose affinity columns, washed and eluted with 10 mM maltose. The
amount of MBP-QD conjugate that eluted was compared by measuring the resultant PL.
In addition, one solution of 560 QD-20 MBP-5HIS was applied to an amylose column
and then washed with 250 mM imidazole while bound. Since imidazole is an analog of
the ring structure found on the histidine residues, this portion of the histidine residue
ostensibly coordinates with the QD surface. Excess imidazole will compete with the
engineered oligohistidine present on MBP-5HIS and eventually outcompete the protein
for binding to the QD surface as the imidazole concentration rises. Indeed, this is the
basis for the oligohistidine-based purification of proteins36. If, as we postulate, MBP-
5HIS-Zn(II) coordination on the QD surface is the basis for QD-MBP self-assembly, then
excess imidazole should adversely affect this coordination, and this is what is observed.
Amylose Resin Assay. 1 mL of crosslinked amylose resin (NEB) was added to a small
chromatographic column and washed 2x with buffer (10 mM NaCl, 10 mM Na-
tetraborate buffer, pH 9.55). The QD-MBP conjugate preparation was applied to the
column followed by washing with 2 mL buffer. QD-MBP complex was eluted with 1 mL
of buffer containing 10 mM maltose. If desired, binding and elution of the QD-MBP
conjugate can be monitored with a hand-held UV light. QDs without an MBP coating
bind irreversibly to the chromatographic matrix and will not pass through the column
with or without application of maltose.
0
1x106
2x106
3x106
1 uM β-CD-Cy3.5Equivalent of 10 MBPCy3/QD10 MBP-Cy3 + 1 uM β-CD-Cy3.5+ 10 mM maltose
B.
PL
(AU
)
0
1x106
3x106
4x106530 QD w/ 10 MBP/QDEquivalent 10 MBPCy3530 QD w/ 10 MBPCy3/QD1 uM β-CD-Cy3.510 MBPCy3 + 1 uM β-CD-Cy3.5Assembled sensor 530QD w/ 10MBPCy3/QD-β-CD-Cy3.5
C.
500 550 600 650 700 750
Supplemental Figure 2. 530 QD w/ 10 MBPD95C-Cy3/QD - β-CD-Cy3.5 controls
0
500x103
1x106
2x106
530 QD530 QD w/ 10 MBP/QD+ equivalent of 10 free Cy3 dye/QD
A.
Wavelength nm
Wavelength nm
500 550 600 650 700 7500
200x103
400x103
600x103
β-CD-Cy3.5
530 QD
MBP-Cy3E.
Supplemental Figure 2. 530 QD w/ 10 MB D95C-Cy3/QD - β-CD-Cy3.5 controls
0 Maltose
10 mM maltose
500 nM maltose
1 µM maltose
5 µM maltose
PL
(AU
)
0
1x106
2x106
530 QD w/ 10 MBP/Dot
530 QD
1 µM β-CD-Cy3.5
530 QD w/ 10 MBP/QD+1 µM β-CD-Cy3.5+ 5 mM maltose
D.
500 550 600 650 7000
500x103
1x106
2x106
2x106 530 QD
MBP-Cy3
Wavelength nm
F.
Time (ns)0 2 4 6 8 10
Nor
mal
ized
inte
nsity
(AU
)
0.0
0.2
0.4
0.6
0.8
1.010MBPCy3 τ = 1.4 +/- 0.1 ns
530QD w/ 10MBPCy3/QD τ = 2.2 ns +/- 0.0 ns
530QD w/ 10MBPCy3/QD + β-CD-Cy35τ = 1.9 +/- 0.1 ns
G.
PL
(AU
)
Supplemental Figure 2. In order to investigate the FRET process and to determine the
direct PL contribution from each individual nanosensor assembly component, a series of
controls was carried out. (A) 530 QD-10MBP-Cy3-β-CD-Cy3.5 controls. Excitation
was at 450 nm (approximate Cy3 absorption minimum). (A) Emission spectra from 530
QDs, 530 QDs conjugated with 10 MBP/QD, and the equivalent concentration of 10 free
Cy3 dye/QD. (B) MBP-Cy3-β-CD-Cy3.5 controls. Emission spectra from the equivalent
amount of 10 MBP monolabeled with Cy3 (molar amount added to 30 pmol of QD for
10MBP/QD), 1 µM β-CD-Cy3.5, the 10 MBP-Cy3 bound to 1 µM β-CD-Cy3.5, and 10
MBP-Cy3 bound to 1 µM β-CD-Cy3.5 after the addition of 5 mM maltose. Note the
almost negligible change in MBP-Cy3 to β-CD-Cy3.5 PL ratio due to excitation at the
Cy3 absorption minimum. (C) 530 QD-MBP-Cy3-β-CD-Cy3.5 sensor assembly.
Emission spectra from 530 QDs conjugated with 10 MBP/QD, the equivalent amount of
10 MBP-Cy3, 530 QDs conjugated with 10 MBP-Cy3/QD, 1 µM β-CD-Cy3.5, 10 MBP-
Cy3 bound to 1 µM β-CD-Cy3.5, and the final assembled sensor, 530QD-10MBP-Cy3-
β-CD-Cy3.5. Note the dramatic loss in QD PL when conjugated with 10 MBP-Cy3 and
the systematic increase in β-CD-Cy3.5 PL. (D) One-step “direct” 530QD-10MBP-β-CD-
Cy3.5 assembly. 530 QDs were conjugated with 10 unlabeled MBP/QD and allowed to
bind with 1 µM β-CD-Cy3.5. The resulting assembly was exposed to 5 mM maltose.
(The arrow points to 1 µM β-CD-Cy3.5 PL and shows the emission of that moiety
measured alone overlapping the sensor + maltose spectrum). Note the negligible
changes and similarity between final sensor + maltose and free 1 µM β-CD-Cy3.5 PL. A
control experiment where 530 QDs conjugated with 10 unlabeled MBP/QD was exposed
to 5 mM maltose and then 1 µM β-CD-Cy3.5 yielded essentially the same result (Data
not shown). (E) Titration of the 530 QD-MBP-Cy3-β-CD-Cy3.5 sensor assembly with
maltose after the direct excitation contributions of MBP-Cy3 and β-CD-Cy3.5 are
subtracted. This leaves only the FRET portion of the sensor assembly titration data. (F)
Data from Figure 4C showing 530 QD and MPBCy3 data deconvoluted with direct
excitation of MBP-Cy3 corrected. (G) Fluorescence lifetime decay curves and τ values
of the Cy3 dye from the 10MBPCy3, 530 QD-10MBP-Cy3 and 530 QD-10MBP-Cy3-β-
CD-Cy3.5 sensor assemblies.
Supplemental Figure 3. β-CD-dye derivative structures25. (A) β-CD-QSY9. (B) β-CD-
Cy3.5. Not to scale. Molecular weights were verified by mass spectral analysis.
Supplemental Table I. Test of QD–MBP sensor specificity using the 560QD-10MBP-
β-CD-QSY9 assembly. The QD-sensor was assembled as described in Methods and
diluted into 3 mL borate buffer in a cuvette for assaying. Two concentrations of each
indicated sugar were added to the cuvette, 100 µM and 1 mM. The ability of each of the
sugars to displace the β-CD-QSY9 from the MBP binding pocket was monitored,
providing a test of MBP recognition specificity in this sensor configuration. The change
(∆) in PL, if any, upon sugar addition was monitored at the QD emission maximum. Any
sugar induced change in PL is reported in the table as the % change in PL compared to
the sensor assembly PL prior to sugar addition. The type and saccharide linkage of each
sugar tested is also described on the table. Soluble MBP (i.e., uncomplexed protein)
specifically binds sugars that contain an α-1,4 glucosidic linkage31. As demonstrated in
the table, only those sugars with this type of saccharide linkage are able to displace β-
CD-QSY9 and increase QD PL. These assays demonstrate that MBP coordinated to QDs
retains its sugar binding specificity.
Supplemental Table II. Estimated energy transfer efficiencies for indicated donor-
acceptor pairs in the sensor assemblies. Efficiencies were estimated in two ways. First,
the direct donor PL loss as described in Methods26 was used in calculations of E.
However, since this does not account for quenching of the donor by other non-FRET
radiative and non-radiative decay processes, FRET efficiencies were also calculated from
direct-excitation-corrected acceptor PL data where possible. Where the acceptor was a
QSY9 dark quencher, an average FRET efficiency based on the other derived data was
used for estimation purposes. However, since the J(λ) and 560 QD-QY is better here,
overall FRET efficiency will probably be higher than estimated for the 560QD-QSY9
pair.
Estimated FRET Relay Efficiency34
Efficiency: D
ADc F
FE =
Where FD is the relative fluorescence of the donor in the absence of acceptor and FAD is
the fluorescence of the acceptor in the presence of donor and corrected for the direct
excitation of the acceptor and any other non-FRET contribution from the donor as
described in Methods.
The FRET process in the 530 QD-10 MBPCy3-β-CD-Cy3.5 assembly can be described
as:
E13
530QD 10 MBPCy3 β-CD-Cy3.5 (1) E12 (2) E23 (3)
Where Eβ-CD-Cy3.5 = E12 * E23 + E13
= 71% * 20% + 6%
= 20%
Eβ-CD-Cy3.5 is the estimated energy β-CD-Cy3.5 receives (FRET only) when bound in the
saccharide pocket of the 530 QD-10 MBPCy3 assembly. The values used are derived
from data such as that in supplementary Figures 2A-C and are all corrected for direct
excitation components.
Supplementary Table I. Quantum Dot - MBP Sensor Assembly Specificity (560QD-10MBP-β-CD-QSY9) Sugar D-Arabinose β-Cyclodextrin D-Galactose D-Glucose D-Mannitol Maltose Sucrose Starch Type1 Mono Oligo (7-monomers) Mono Mono Mono Di Di Oligo
Oligosaccharide Linkage
NA α-1,4 glucosidic
NA NA NA α-1,4 glucosidic
α-1, β-2 glucosidic
α-1,4 glucosidic
∆ PL2 100 uM < 1% 47% < 1% < 1% < 1% 96% < 1% 55%
∆ PL2 1 mM < 1% 54% < 1% < 1% 1.5% 104% 1.6% 58% 1Mono, Di and Oligo – saccharide 2∆ PL = Percentage increase in PL intensity with respect to the initial value of the nanosensor, upon addition of the indicated amount of sugar NA-Not applicable Supplementary Table II. Estimated Energy Transfer Efficiency of Sensor Assemblies
Donor Acceptor Estimated Efficiencies from Donor ∆PL
Estimated Efficiencies Corrected from Acceptor ∆PL
Comments
530 QD + 10 MBP/QD 1 uM β-CD-Cy3.5 ~ 13% ~ 6% MBP adhered to QD surface 530 QD 10 MBP-Cy3/QD ~ 94% ~ 71% MBP-Cy3 adhered to QD surface
0.1 uM MBP-Cy3 1 uM β-CD-Cy3.5 ~12% ~20%* No QD present 530 QD + 10 MBP-Cy3/QD 1 uM β-CD-Cy3.5 ~ 25% ~ 20% MBP-Cy3 adhered to QD surface
560 QD + 10 MBP/QD 1 uM QSY9 free dye ~ 7.5% < 6 % MBP adhered to QD surface 560 QD + 10 MBP/QD 1 uM β-CD-QSY9 ~ 47% ~ 35% MBP adhered to QD surface
λ Excitation = 450 nm The amount of QD used was 30 pmol. per experiment26. *The low value is due to exciting close to a Cy3 absorption minima