A R T I C L E S
Martin et al.
PAMAM exhibited short large amplitude events suggesting
significant changes in the polymer structure. The recordings,
which were performed in another study,24 displayed events
characterized by a current blockade of 51 ( 3%, an average
duration of 14 ( 2 ms, and a frequency of occurrence of 0.2 (
structurally defined membrane pore RHL, dendrimers exhibited
a sharp size-dependent permeation cutoff and readily identified
a 2.9 nm wide pore entrance. PAMAM-PDP dendrimers with
diameters from 2 to 10 nm are well suited to probe various
nanometer-sized pore-forming proteins with important biomedi-
-
1
39
0
.02 s . The short events originated from the threading of the
cal roles such as bacterial pore-forming toxins, pores of the
free end of the polymer chain into a narrow constriction of the
complement system, or dilating purinergic receptors.40 In
addition, the approach can also be applied to explore the
molecular structure of interesting biological nanomaterials such
pore through the â-barrel toward the trans side of the pore
4,25,31
(
Figure 1B).2
Our single-molecule data therefore offer a
3
8,41
glimpse of the different structural dynamics of individual “hard”
positively charged PAMAM dendrimers as compared to “soft”
neutral PEG chains.
as porous S-layer proteins.
The study presents a new way to alter the properties of
proteins via targeted chemical modification with hyperbranched
dendrimers. Coupling of nonbranched linear organic polymers
or biopolymers has been used in the past to modify or expand
the natural characteristics of proteins such as in pharmacology
to increase the half-life of therapeutic proteins via PEGylation,42
in molecular biology to introduce sequence specificity into
nucleases via attachment of a DNA oligonucleotides,43 or in
microarray technology to achieve targeted immobilization of
Discussion
This report describes the generation of sulfhydryl-reactive
PAMAM dendrimers and their coupling to cysteine mutants of
a protein pore of known structure. The extent of reaction of the
dendrimers was found to depend on the PAMAM generation
and position of the engineered residues in the protein pore. G5
with a hydrodynamic diameter of 6.2 nm did not enter the cis
entrance, while G3 with 4.2 nm coupled inside the pore. This
clear cutoff in the permeation properties is in contrast to the
greater accessibility of flexible PEG chains. For example, PEG-
MAL 10 and 5 kD with comparable hydrodynamic diameters
of 6.2 and 4.4 nm35 coupled inside the pore lumen. The results
of the present study are in line with another report on the use
of PEG. Using the same RHL pore as a model system it was
found that linear PEG polymers 1-3 kD (hydrodynamic
diameters of 2.1-3.0 nm) could pass through the 1.3 nm wide
inner constriction of RHL.27 Only PEG 5 kD with a diameter
of 4.4 nm translocated poorly. Hence, a PEG chain was only
restricted in its permeation when the hydrodynamic parameter
was at least 3.5 times higher than the pore constriction. By
contrast, this value is 2 for G5-PAMAM and expected to be
even lower for higher generation dendrimers with more rigid
structure.
4
4
PNA-modified proteins onto DNA-microarrays. While den-
drimers have been attached to proteins to produce new types
of immunoreagents with enhanced binding capacity,45 their use
to alter the conductance properties of pores is new. Our results
show that a dendrimer molecule filled at least the lumen of the
engineered pore and thus regulated the ion flux.
The hyperbranched dendrimers were demonstrated to function
as ion-selectivity filters for the passage of small ions through
the pore. As expected, the positively charged primary amines
on the surface of the PAMAM dendrimer led to a preferred
permeation of anions over cations. The advantage of PAMAM
over other ion-selective elements such as cyclodextrins32 is that
the dendrimer filters are available in different sizes up to 10
nm. This offers the possibility to use dendrimers for inorganic
pores with wide lumen. PAMAM was also demonstrated to
function as a molecular sieve to control the passage of molecules
through the pore based on their molecular weight. Our results
show that the RNA oligonucleotides were blocked while smaller
ions were only minimally affected. It is expected that the
molecular weight cut off of the molecular sieves can be tuned
by increasing or decreasing the size of the dendrimer or changing
its chemical composition. While movement of RNA through
G3 PAMAM-filled pores is blocked, use of smaller dendrimerss
in combination with higher potentialsscould possibly enable
passage of nucleic acids at reduced translocation speeds with
The sulfhydryl-reactive dendrimers presented in this study
are a new type of research reagent for examination of the
molecular structure of proteins. The reagents can be used in
2
6
the substituted-cysteine accessibility method (SCAM), which
infers the surface accessibility of residues by determining how
fast sulfhydryl-reactive reagents couple to single-cysteine
mutants of a protein. SCAM has been widely exploited to probe
the structure of membrane proteins and ion channels in
2
6,36
46
combination with patch clamp or lipid bilayer recordings,
applications in DNA sensing.
and gel electrophoretic mobility shift assays.24,27,37,38 Despite
the wide range of sulfhydryl-active organic reagents or poly-
Our approach to alter pore permeation properties by placing
a spherical dendrimer inside the lumen is new. Use of hyper-
branched dendrimers has specific advantages over other spheri-
2
7
meric reagents, most are too small or too flexible for
membrane proteins with pore diameters of more than 2-3 nm
because they can either access all pore-lining residues or their
coupling to cysteine residues does not give rise to an appreciable
current block or gel shift, making it difficult to detect successful
modification. Sulfhydryl-reactive PAMAM dendrimers with
diameters of several nanometers can overcome these constraints
as shown in this study. In calibration experiments with the
(
(
(
39) Parker, M. W.; Feil, S. C. Prog. Biophys. Mol. Biol. 2005, 88, 91-142.
Menestrina, G. Pore-forming peptides and protein toxins; Taylor & Francis,
CRC: Boca Raton, FL, 2003.
40) Surprenant, A.; Rassendren, F.; Kawashima, E.; North, R. A.; Buell, G.
Science 1996, 272, 735-738. Khakh, B. S.; North, R. A. Nature 2006,
442, 527-532.
41) Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M. Angew. Chem., Int. Ed. 1999,
38, 1035-1054.
(42) Veronese, F. M.; Pasut, G. Drug. DiscoVery Today 2005, 10, 1451-1458.
(43) Corey, D. R.; Schultz, P. G. Science 1987, 238, 1401-1403.
(44) Winssinger, N.; Harris, J. L.; Backes, B. J.; Schultz, P. G. Angew. Chem.,
Int. Ed. Engl. 2001, 40, 3152-3155.
(
35) Scherrer, R.; Gerhardt, P. J. Bacteriol. 1971, 107, 718-735.
(
36) Akabas, M. H.; Stauffer, D. A.; Xu, M.; Karlin, A. Science 1992, 258,
(45) Singh, P. Bioconjugate Chem. 1998, 9, 54-63. Patri, A. K.; Myc, A.; Beals,
J.; Thomas, T. P.; Bander, N. H.; Baker, J. R., Jr. Bioconjugate Chem.
2004, 15, 1174-1181. Kobayashi, H.; Kawamoto, S.; Saga, T.; Sato, N.;
Ishimori, T.; Konishi, J.; Ono, K.; Togashi, K.; Brechbiel, M. W.
Bioconjugate Chem. 2001, 12, 587-593.
3
07-310.
(37) Walker, B.; Bayley, H. J. Biol. Chem. 1995, 270, 23065-23071. Lu, J.;
Deutsch, C. Biochemistry 2001, 40, 13288-13301.
(38) Howorka, S.; S a´ ra, M.; Wang, Y.; Kuen, B.; Sleytr, U. B.; Lubitz, W.;
Bayley, H. J. Biol. Chem. 2000, 48, 37876-37886.
(46) Bayley, H. Curr. Opin. Biotechnol. 2006, 10, 628-637.
9648 J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007