Assembly of nanoparticle-protein binding complexes: From monomers to ordered arrays

Article abstract of DOI:10.1002/anie.200701180

(Figure Presented) Tag it: Well-defined monomers, dimers, trimers, three-dimensional spherical shells, and two-dimensional ordered arrays of genetically engineered proteins were constructed by using functionalized gold nanoparticles. Nanoparticle size, fu

Full text of DOI:10.1002/anie.200701180

Angewandte
Chemie
DOI: 10.1002/anie.200701180
Nanoparticle–Protein Assembly
Assembly of Nanoparticle–Protein Binding Complexes: From
Monomers to Ordered Arrays**
Minghui Hu, Luping Qian, Raymond P. Briæas, Elena S. Lymar, and James F. Hainfeld*
Nanoparticle (NP)–biomolecule binding complexes have
shown promising applications in imaging, sensing, catalysis,
electronics, and cell targeting because NPs possess size-
dependent optical, electrical, and magnetic properties while
biomolecules can perform unique biological functionalities.[1]
NP–biomolecule hybrid structures with various architectures
have been constructed by using biomolecules as templates.[2]
Meanwhile, the site-specific recognition has been extensively
employed for many applications that require biomolecule
structural control.[3] Although biomolecules themselves show
versatile assembling properties,[4] NPs have been used as
templates to construct NP–biomolecule binding complexes
through interactions with DNA,[5] proteins,[6] and viral
capsids.[7] However, the potential of functionalized NPs as
templates to assemble proteins has not been fully explored,
and the control of NP–protein nanostructures remains
challenging.
tion with a solution of NiCl2 to form Ni-NTA Au NPs.We
used these NPs as templates to assemble two 6 ” His-tagged
proteins, an adenovirus serotype 12 (Ad12) knob,[10] and a
mycobacterium tuberculosis (Mtb) 20S proteasome.[11] In the
60-kDa C3-symmetric knob protein, a 6 ” His tag was fused
onto the N terminus of each subunit, forming a highly
localized binding motif (Figure 1a).The 750-kDa D7-sym-
Herein, we report the formation of NP–protein hybrid
complexes with controlled geometry, stoichiometry, orienta-
tion, and specificity by tailoring the sizes of binding NPs and
placement of genetic tags in proteins.Well-defined NP–
protein complexes are formed through site-specific binding
between 6 ” histidine (His) tags in proteins and nickel-
nitrilotriacetic acid (Ni-NTA) functional groups on Au NPs
with sizes ranging from 1 to 4 nm.Our study demonstrates
that NPs are not only appealing templates for assembling
functional biomolecules, but also provide a mortar to
construct novel geometrical and topological architectures of
hybrid NP–protein complexes.
In this study, we synthesized a ligand-containing NTA
moiety, (1S)-N-[5-[(4-Mercaptobutanoyl)amino]-1-carboxy-
pentyl]iminodiacetic acid (NTA-Lys-SH) (see Scheme S1 in
the Supporting Information).[8] This ligand was used to
synthesize 1.3-nm NTA Au NPs from Au(PPh3)8Cl3 (see
Scheme S2 and Figure S1 in the Supporting Information)[9]
and 4.4-nm NTA Au NPs from HAuCl4 (see Scheme S3 and
Figure S2 in the Supporting Information) followed by reac-
Figure 1. 3D structures of Ad12 knob and 20S proteasome proteins
showing the binding motif to Au NPs. a) Knob protein[10] in which a
6”His tag (indicated by the arrow) is attached to the N terminus of
each subunit; b) proteasome protein[11] in which a 6”His tag (indi-
cated by the arrow) is attached to the C terminus of each of the
fourteen b subunits around the central equator.
metric proteasome protein has a stack of four rings, abba, and
each layer has seven identical subunits.A 6 ” His tag was
fused onto the C terminus of each b subunit, thus creating
seven pairs of binding motifs that are uniformly dispersed
around the periphery of the two central layers (Figure 1b).
For the Ad12 knob, the close proximity of the three 6 ”
His tags favored the formation of monomers, dimers, trimers,
and three-dimensional (3D) spherical shells on Au NPs with
sizes ranging from 1 to 4 nm.The knob protein solution was
mixed with 1.3-nm Au NPs, followed by separation by using
size-exclusion chromatography (SEC; Figure 2).Two major
fractions at 32.0 and 37.4 min were collected, both of which
showed the absorption characteristics of 1.3-nm Au NPs
(continuously from 200–500 nm) and proteins (280 nm; see
Figure S4 in the Supporting Information).High-angle annular
dark field (HAADF) scanning transmission electron micro-
[*] Dr. M. Hu, L. Qian, Dr. R. P. Briæas, Dr. E. S. Lymar, Dr. J. F. Hainfeld
Biology Department
Brookhaven National Laboratory
Upton, NY 11973 (USA)
Fax: (+1)631-3443407
E-mail: hainfeld@bnl.gov
[**] This work was supported by BNL LDRD Grant 04-055, DOE Grant
06742, and NIH Grants P41EB002181 and R01RR017545. We thank
Y. Zhang, P. I. Freimuth, G. Hu, H. Li, and C. F. Nathan for kindly
providing protein samples, and J. S. Wall, M. Simon, B. Lin, and F.
Kito for assistance with STEM analysis.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Figure 2. Size-exclusion-chromatography profile of binding complexes
of 6”His-tagged Ad12 knob proteins and 1.4-nm Ni-NTA Au NPs
showing separation of the monomer and dimer (occasional trimer).
A
280 =absorbance at 280 nm.
scopy (STEM) analysis indicated that the fraction at 32 min
was protein monomers on Au NPs with an average size of
0.8 nm (Figure 3a,d), whereas the fraction at 37 min was
mainly protein dimers on Au NPs with an average size of
1.6 nm (Figure 3b,e). A few trimers on larger NPs were found
in the dimer fraction (Figure 3b,f).When 44.-nm Au NPs
were used, 3D NP–protein core-shell complexes were formed
instead of discrete numbers of the protein (Figure 3c,g).In
the presence of excess proteins, all of the Au NPs were
encapsulated by proteins, showing a high binding efficiency.
This structural dependence on the NP size appears to result
from the surface area available for protein binding.[5b,6a]
For the 20S proteasome, the dispersed distribution of 6 ”
His tags resulted in the formation of two-dimensional (2D)
ordered tetragonal arrays with a 1:1 stoichiometry instead of
3D spherical shells.Here, we used 37.-nm Au NPs with a
narrower size deviation of 0.7 nm, which were isolated by
centrifugation of 4.4-nm NPs (see Figure S3 in the Supporting
Information).The formation of ordered tetragonal arrays
over several hundred nanometers has been observed (Fig-
ure 4a).As predicted from the position of 6 ” His tags in the
proteasome protein (Figure 1b), NPs bound exactly to the
central equator of cylindrical proteasome (Figure 4b,c).Study
in situ by using cryogenic (cryo)-TEM confirmed the forma-
tion of tetragonal NP–protein arrays in the solution
(Figure 5).The size of Au NPs in the arrays had a lower
deviation than that of as-used arrays (see Figure S3 in the
Supporting Information).This could result from size selection
during the array growth in which more identical NPs are
incorporated into the arrays.An averaged image over multi-
ple protein molecules in an array revealed a top view of the
cylindrical structure of 20S proteasomes.This architectural
formation can be understood from the steric restriction based
on the Paulingꢀs rules (see Table S1 in the Supporting
Information).[12]
Figure 3. TEM images of monomers, dimers, trimers, and core-shell
assemblies of Ad12 knob proteins and Au NPs with different sizes.
HAADF STEM images of a) monomer and b) dimer/trimer of 6”His-
tagged Ad12 knob proteins attached to 1.4-nm Au NPs, which were
isolated by using size-exclusion chromatography; c) negative-stain
TEM image of core-shell binding complexes of knob proteins with 4.4-
nm NPs; d)–g) close-up viewof monomers, dimers, trimers, and 3D
spherical shells, respectively, in which each knob molecule is marked
by an arrow.
Taking the Ad12 knob and Mtb 20S proteasome proteins as
examples, monomers, dimers, trimers, 3D spherical shells, and
2D ordered arrays are formed.These hybrid structures
formed by coupling NPs and proteins are promising for
novel applications.The knob protein is typically found on the
end of viral spikes and mediates adenovirus attachment and
uptake through a high binding affinity with the coxsackie and
adenovirus receptor (CAR) on the surface of many cell
types.[10] The NP–knob binding complexes potentially enable
visualization of cellular attachment and nanoparticle delivery.
The proteasome protein is involved in mycobacterial defence
When 6 ” His tags were removed from a thrombin
cleavage site in Ad12 knob and 20S proteasome proteins,
these proteins showed no binding to Ni-NTA Au NPs
(Figure 6).This is indicative of the highly specific interactions
between these Au NPs and proteins.
These results demonstrate that NP–protein binding com-
plexes can be constructed with predictable architecture by
tailoring NP size, functionality, and placement of genetic tags.
5112
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Figure 6. Negative-stain TEM images of Au NPs with a) Ad12 knob
and b) 20S proteasome proteins whose 6”His tags were removed and
therefore showed no formation of binding complexes.
Keywords: electron microscopy · hybrid materials ·
.
nanoparticles · proteins · self-assembly
[1] a) L.Montesano-Roditis, D.G.Glitz, R.R.Traut, P.L.Stewart,
J. Biol. Chem. 2001, 276, 14117 – 14123; b) Y.Xiao, F.Patolsky,
E.Katz, J.F.Hainfeld, I.Willner, Science 2003, 299, 1877 – 1881;
c) L.Bumba, M.Tichy, M.Dobakova, J.Komenda, F.Vacha,
J.
Struct. Biol. 2005, 152, 28 – 35; d) C.You, S.S.Agasti, M.De,
M.J.Knapp, V.M.Rotello, J. Am. Chem. Soc. 2006, 128, 14612 –
14618; e) R.J.Tseng, C.Tsai, L.Ma, J.Ouyang, C.S.Ozkan, Y.
Yang, Nat. Nanotechnol. 2006, 1, 72 – 77; f) D.C.Deniger, A.A.
Figure 4. Negative-stain TEM images of binding complexes of 3.7-nm
Au NPs and 20S proteasome proteins. a) NP–proteasome 2D arrays;
b) top and c) side viewof a proteasome protein bound to an Au NP in
which the two innersubunit b rings are indicated by the arrows.
Kolokoltsov, A.C.Moore, T.B.Albrecht, R.A.Davey,
Lett. 2006, 6, 2414 – 2421.
Nano
[2] a) M.Allen, D.Willits, J.Mosolf, M.Young, T.Douglas,
Adv.
Mater. 2002, 14, 1562 – 1565; b) D.Ishii, K.Kinbara, Y.Ishida, N.
Ishii, M.Okochi, M.Yohda, T.Aida, Nature 2003, 423, 628 – 632;
c) R.Djalali, Y.Chen, H.Matsui, J. Am. Chem. Soc. 2002, 124,
13660 – 13661; d) R.A.McMillan, C.D.Paavola, J.Howard,
S.L.Chan, N.J.Zaluzec, J.D.Trent,
Nat. Mater. 2002, 1, 247 –
252; e) A.S. Blum et al., Nano Lett. 2004, 4, 867 – 870; f) R.
Baron, B.Willner, I.Willner, Chem. Commun. 2007, 323 – 332.
[3] a) K.K. Caswell, J.N. Wilson, U.H.F. Bunz, C.J. Murphy,
Am. Chem. Soc. 2003, 125, 13914 – 13915; b) C.M.Niemeyer,
W.Burger, J.Peplies, Angew. Chem. 1998, 110, 2391 – 2395;
Angew. Chem. Int. Ed. 1998, 37, 2265 – 2268; c) J.F.Hainfeld, W.
Liu, C.M.R.Halsey, P.Freimuth, R.D.Powell, J. Struct. Biol.
J.
1999, 127, 185 – 198; d) M.Aubin, D.G.Morales, K.Hamad-
Schifferli, Nano Lett. 2005, 5, 519 – 522.
[4] a) T.O.Yeates, J.E.Padilla, Curr. Opin. Struct. Biol. 2002, 12,
464 – 470; b) P.Ringler, G.E.Schulz,
109; c) L.Nasalean, S.Baudrey, N.B.Leontis, L.Jaeger,
Acids Res. 2006, 34, 1381 – 1392; d) J.C.T.Carlson, S.S.Jena, M.
Flenniken, T.Chou, R.A.Siegel, C.R.Wagner, J. Am. Chem.
Soc. 2006, 128, 7630 – 7638; e) K.Sugimoto, S.Kanamaru, K.
Iwasaki, F.Arisaka, I.Yamashita, Angew. Chem. 2006, 118,
Science 2003, 302, 106 –
Nucleic
Figure 5. Cryo-TEM images of tetragonal arrays of 3.7-nm Au NPs and
20S proteasome proteins with a stoichiometry of 1:1. The inset shows
an averaged image over multiple protein molecules in an array.
2791 – 2794; Angew. Chem. Int. Ed. 2006, 45, 2725 – 2728; f) S.
Ghosh, M.Reches, E.Gazit, S.Verma, Angew. Chem. 2007, 119,
2048 – 2050; Angew. Chem. Int. Ed. 2007, 46, 2002 – 2004.
[5] a) F.Patolsky, Y.Weizmann, O.Lioubashevski, I.Willner,
Angew. Chem. 2002, 114, 2429 – 2433; Angew. Chem. Int. Ed.
2002, 41, 2323 – 2327; b) CJ..Ackerson, MT. .Sykes, RD. .
Kornberg, Proc. Natl. Acad. Sci. USA 2005, 102, 13383 – 13385;
against host nitrosative stress.[11] The enzyme activity may be
modulated upon binding to Au NPs.The formation of 2D NP–
protein arrays might improve alignment and enhance reso-
lution in cryo-electron microscopy structural studies.
c) S.J.Hurst, A.K.R.Lytton-Jean, C.A.Mirkin,
Anal. Chem.
2006, 78, 8313 – 8318.
[6] a) A.A.Vertegel, R.W.Siegel, J.S.Dordick,
Langmuir 2004, 20,
6800 – 6807; b) F.Patolsky, Y.Weizmann, I.Willner, Nat. Mater.
2004, 3, 692 – 695; c) S.Srivastava, A.Verma, B.L.Frankamp,
V.M.Rotello, Adv. Mater. 2005, 17, 617 – 621.
Received: March 17, 2007
Published online: May 30, 2007
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[7] a) L.Loo, R.H.Guenther, V.R.Basnayake, S.A.Lommel, S.
[10] MC. .Bewley, K.Springer, Y.Zhang, P.Freimuth, JM. .
Flanagan, Science 1999, 286, 1579 – 1583.
[11] G.Q.Hu, G.Lin, M.Wang, L.Dick, R.Xu, C.Nathan, H.L.Li,
Mol. Microbiol. 2006, 59, 1417 – 1428.
Franzen, J. Am. Chem. Soc. 2006, 128, 4502 – 4503; b) C.Chen
et al., Nano Lett. 2006, 6, 611 – 615.
[8] O.Du Roure, C.Debiemme-Chouvy, J.Malthete, P.Silberzan,
Langmuir 2003, 19, 4138 – 4143.
[12] L.Pauling, J. Am. Chem. Soc. 1929, 51, 1010 – 1026.
[9] G.H.Woehrle, M.G.Warner, J.E.Hutchison,
J. Phys. Chem. B
2002, 106, 9979 – 9981.
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