Biology Reference
In-Depth Information
measured forces, and to attach the biomolecules at a low surface density to
ensure single-molecule detection.
A good strategy to covalently anchor proteins on tips is to use a
polyethylene glycol (PEG) crosslinker which provides motional freedom and
prevents denaturation. Tips are irst modiied with amino groups, further
reacted with PEG linkers carrying benzaldehyde functions that are then
directly attach to proteins through their lysine residues.
Another approach
is to use self-assembled monolayers of alkanethiols on gold tips. 18,19 Both
methods make it possible to orientate the attached biomolecules via their
C-terminal or N-terminal domains by linking recombinant histidine-tagged
proteins onto tips coated with nitrilotriacetate groups. 18,24
22,23
15.2.2 The NTA
His 6 System: A Powerful Plaorm for SMFS
The site-directed nitrilotriacetic acid (NTA)-polyhistidine (His n ) system
has recently emerged as a powerful platform for SMFS studies. The NTA-
His 6 binding chemistry, well known for afinity puriication of recombinant
proteins, involves the formation of a hexagonal complex between the
tetradental ligand NTA and divalent metal ions like Ni 2+ . Since NTA occupies
four of the six coordination sites of Ni
-
, the two remaining sites are
accessible to other Lewis bases, e.g., the histidines of tagged proteins. In
the SMFS context, the NTA-His 6 approach offers the important advantage
that the attached proteins remain fully functional and can be oriented
via their C-terminal or N-terminal His tag. A pertinent question though is
to ensure that the NTA-His bond is suficiently strong for stable protein
immobilization during force measurements. This issue was recently
clariied by recording force-distance curves between AFM tips modiied
with Ni
2+
-NTA-terminated alkanethiols and solid supports functionalized
with His 6 -Gly-Cys peptides ( Fig. 15.1 ) . 25 Consistent with the earlier work of
Kienberger
2+
the adhesion force histogram showed three maxima at
rupture forces of 153 ± 57 pN, 316 ± 50 pN and 468 ± 44 pN, attributed
to monovalent and multivalent interactions between a single His 6 moiety
and one, two and three NTA groups, respectively ( Fig. 15.1 ) . The plot of
adhesion force versus log of the loading rate revealed a linear regime, from
which a kinetic off-rate constant of dissociation was deduced. The obtained
value was in the range of that estimated for the multivalent interaction
involving two NTA, using luorescence measurements, and may account for
an increased binding stability of the NTA-His 6 bond. Since the measured
forces of ~150 pN are well above the 50-100 pN unbinding forces typically
observed for receptor-ligand pairs, it was concluded that the NTA-His 6
system is a well-suited platform for the stable, oriented immobilization of
proteins in SMFS studies.
et al.
,
24
 
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