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M it i g P

Monitoring
Protein
t i St
Structure
t
on the
th Surface
S f
off Gold
G ld Nanoparticles
N
p ti l using
i g NMR Spectroscopy
Sp t
py
Ailin Wang,
Wang
g, Karen Woods
Woods,, Tam Vo
Vo,, Alex Coats,
Coats, and Nicholas C
C. Fitzkee
D
Department
t
t off Chemistry,
Chemistry
Ch i t Mississippi
Mi i i i State
St t University
U i
it
Abstract

R
Results
l

Because of their unique


q
spectroscopic
p
p p
properties
p
and biocompatibility,
biocompatibility
p
y
protein-functionalized
protein
t i functionalized
f
ti
li d gold
ld nanoparticles
ti l
(A NP ) have
(AuNPs)
h
many potential
t ti l
diagnostic
g
and therapeutic
p
applications
applications.
pp
Surface bound p
Surface-bound
proteins can be used
b th as molecular
both
l
l sensors and
d drug
d
d li
delivery
vectors,
t
and
d the
th plasmonic
l
i
properties
p
p
of g
gold enable the detection of veryy small changes
g
in surface
chemistry.
h i t
U f t
Unfortunately,
t l the
th design
d i
off general
general-purpose,
l purpose, functionalized
f
ti
li d
AuNPs is severelyy complicated
p
byy our limited understanding
g of p
protein
structure
t t
on nanoparticle
ti l surfaces.
f
S
Some
enzymes remain
i active
ti on AuNPs
A NP
while others are inactive,
inactive and it is currentlyy impossible
p
to p
predict which
b h i
behavior
will
ill be
b observed.
b
d To
T address
dd
thi problem,
this
bl
we have
h
recently
tl
developed
p
several new NMR-based
NMR based approaches
pp
for monitoring
g p
protein
structure
t t
on AuNP
A NP surfaces.
f
W find
We
fi d that
th t the
th adsorption
d
ti
capacity
it off 15 nm
AuNPs can be p
predicted using
g the native structure,
structure suggesting
gg
g that p
proteins
remain
i globular
l b l on the
th AuNP
A NP surface.
f
Additi
Additionally,
ll we demonstrate
d
t t that
th t
proteins can be displaced
p
p
from AuNPs byy organothiols,
organothiols
g
supporting
pp
g a case for
reversible
ibl binding.
bi di
Fi ll we have
Finally,
h
used
d hydrogen/deuterium
h d
/d t i
exchange
h
((HDX)) to monitor structural p
perturbations of two model p
proteins GB3 and
proteins,
Ubi iti when
Ubiquitin,
h
b
bound
d to
t AuNPs.
A NP We
W find
fi d no significant
i ifi
t changes
h
i slow
in
l
HDX rates ((5-300
(5 300 min),
min)) suggesting
gg
g that AuNP-induced
AuNP induced structural changes
g
are smallll for
f these
th
t
two
proteins.
t i
T
Together,
th
th
these
results
lt supportt a model
d l
where most of a p
proteinss native contacts are p
protein
preserved upon
p
adsorption
adsorption,
p
although
lth
h larger
l
changes
h
may occur over long
l
ti
timescales.
l

Q
Quantification
Qua
ttificattio by
b NMR

Is Binding
Is
Bi d
di g Reversible?
Re e sibl
b e?

Fig
Figure
6 Diagram
6:
Di g
off p
protein-AuNP
t i A NP bi
binding
di g model.
d l

Fig
Figure
4 Bound
4:
B
d protein
p t i levels
l
l in
i the
th presence/absence
p
/ b
off MBI.
MBI
Figure 2: (a) 1D titration of GB3 as AuNPs are added.
added (b) Calibration curve to
calculate adsorption capacity.
capacity

Adsorbed GB3 does not contribute to signal;


reference compound
p
(TMSP)
(
) held at constant
concentration

Bound
B
d protein
t i measured
d after
ft addition
dditi off smallll
organothiol
thi l (2
(2-mercaptobenzimidazole
mercaptobenzimidazole,
t b
i id
l MBI)
Protein levels return to normal;; binding
g can be
di l
displaced
displaced,
d suggesting
ti
reversibility
ibilit

Calibration
C lib ti curves indicate
i di t how
h
much
h
macromolecule adsorbs to AuNP

I t d ti
Introduction
P t i Functionalized
Protein-Functionalized
Protein
F
ti
li d G
Gold
ld Nanoparticles
N
ti l

Th
Three-Step
St p M
Model
d l off Adsorption
Ad
pti

Assumes saturated binding


g (>95%
(
bound))

Reversible
R
ibl association
i ti (kex > 10 ms);
) smallll
perturbations
t b ti
to
t protein
t i structure
t t
Slow reorientation for maximal adsorption;
p
; allows
f close packing
for
p
g on AuNP surface
f
Eventually
E
t ll protein
t i forms
f
irreversible
i
ibl attachments
tt h
t
(e g through thiols)
(e.g.

Real-time
Real
time H
H-D
D Exchange
g

Di
Discussion
i

C
Compact
Protein
P
i on AuNPs
A NP

Using radius of gyration,


gyration it is possible to estimate
occluded surface area and adsorption
p
capacity
p
y for
a folded protein
p

Figure 5: Hydrogen exchange profiles of GB3 (a) and ubiquitin (Ubq,


(Ubq b) with
and
d without
ih
A NP at pH
AuNPs
H 6.5.
6
I the
In
h protein
protein-AuNP
i AuNP
A NP sample,
l 50%
0% off the
h totall
protein
t i was adsorbed
d b d to
t AuNPs.
A NP

Figure
g
1: Possible behaviors of p
proteins adsorbed to AuNPs.
AuNPs Addition of p
protein ((antibody)
y) to
stabilized AuNPs (top) can either: distort protein structure and perturb function (left path),
path) leave
structure unperturbed and retain avidity (central path),
path) or induce aggregation and fibrillation (right
path) Figure taken from Shemetov,
path).
Shemetov A.
A A.,
A et al.
al 1

Proteins
P t i known
k
to
t adsorb
d b spontaneously
t
l to
t gold
ld nanoparticle
ti l
(A NP) surfaces
(AuNP)
f
Unanswered q
questions: ((1)) Are adsorbed proteins
p
folded? (2)
( )
How much protein
p
can bind? (3)
( ) Is adsorption
p
reversible?
Biomedical
Bi
di l Applications:
A li ti
Bi l i l sensors,
Biological
sensors drug
d
delivery,
delivery
d li
cancer treatment2
Several quantification
q
methods exist: proteolytic
p
y cleavage
g with
MALDI TOF MS,
MALDI-TOF
MS 3 absorbance
b b
d t ti after
detection
ft centrifugation,
centrifugation
t if g ti
i t i ll labeled
isotopically-labeled
isotopically
l b l d proteins
t i 4
NMR-based
NMR based hydrogen/deuterium exchange (HDX) enables the
detection of bovine -lactalbumin
lactalbumin ((BLA)) adsorbed on polystyrene
p y y
5
nanospheres
ph

Fi
Figure
3:
3 Predicted
P di t d vs. observed
b
d adsorption
d
ti capacity.
it The
Th inset
i
t shows
h
a closeclose
l
up o
off agreement
ag ee e t for
fo WW and
a d larger
la ge proteins.
p ote
t i s

R id
Residues
RG ()
Ob
Observed
d*
P di d*
Predicted
MBI

1490 40
1490

GSH
3
3.75
1600 100
1600
1600
WW
45
10.52
175 6
175
203
GB3
56
10.62
200 20
200
199
UBQ
76
12.04
156 12
156
155
BCA
259
17 17
17.17
63 10
63
76
BSA
583
26 15
26.15
30 10
30
33
*Reportedasthenumberofmoleculesboundtoasingle15nmAuNP.
Reported as the number of molecules bound to a single 15nm AuNP

Folded protein structure used to calculate radius of


gyration
gy
a o
Predict
P di t adsorption
d pti capacity
p ity using
i g occluded
l d d surface
f
assuming
i spherical
h i l protein/AuNP
t i /A NP6
Excellent agreement observed,
observed implies complete
coverage
g and globular
g
(if
( not folded)) protein
p

Hydrogen
y g exchange
g ((HDX)) is initiated by
y adding
g
D2O and monitored byy a recording
g series of 15NN 1H
HSQC spectra
p t up
p to
t 960 min
i after
ft initiation
i iti ti
Slow
Slo exchange
e change rates are observed
obser ed on the
secondary structure elements
elements. AuNP
AuNP-induced
induced
differences are minor7
Fast
F t exchange
h
rates
t show
h
a similar
i il trend:
t d only
l
minor
i
differences
diff
observed
b
d (data
(d t nott shown)
h
)
Consistent with relatively
y small structural
perturbations on the AuNP surface
p

Acknowledgement.
Acknowledgement
g
We thank Dr.
Dr Ad Bax for g
generouslyy p
providing
g
GB3 p
plasmids used
sed in this study,
st dy, and Dr.
Dr Dongmao
g
Zhang
g for helpful
helpf
p l
di
discussions.
i
Thi work
This
k was supported
t d by
b the
th National
N ti
l Institutes
I tit t
off
Health (NIH) under award R15GM113152.
R15GM113152
The content is solelyy the responsibility
p
y of the
authors and does not necessarilyy represent
p
th views
the
i
off the
th NIH.
NIH

Uniform
U if
HDX pattern
tt
b t
between
protein
t i in
i absence
b
and presence of AuNP is consistent with the
quantification result; stabilized secondary structure
remains native
native-like
like on AuNP
Protein
P t i may harden
h
harden
d on the
th surface
f
off AuNP
A NP
d i th
during
the third
thi d step;
t
structure
t t
may change
h
over
time

R f
References
1. Shemetov
1
Shemetov,
Sh
t A
A. A
A., ett al
al.
l (2012)
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Ju
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Yeo. W
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