binding protein (MBP) was added (Fig. 3a). SrtA is a transpeptidase involved in bacterial cell wall assembly that anchors surface
proteins containing an LPXTG recognition motif to the amino
group of a pentaglycine on the peptidoglycan cell wall [17]. This
enzyme can be used in vitro to covalently link a labeled peptide
containing an N-terminal glycine residue to a protein with a SrtA
recognition site (Fig. 3a). As the MBP is positioned at the
C-terminus of the recognition site, it is cleaved off during the
labeling reaction and it can easily be judged by SDS-PAGE whether
the labeling is completed (Fig. 3c). Following the labeling reaction,
the FITC-labeled clamp loader was isolated by SEC (Fig. 3b, c) and
checked for homogeneity by AUC (Fig. 2).
1.4 Template/
Primer DNA
The sequences of the oligonucleotides were chosen according to
Bloom et al. [18], though omitting the 3
0 -overhang of the template
makes them thereby better comparable to the 105-mer/50 -fluorescinated 50-mer template/primer used in SPR [13]. However, they were designed to have shorter double-stranded regions
since only about ten nucleotides of double-stranded DNA have
been shown to be involved in interactions with the clamp loader
[15]. We used a template/primer consisting of a 30-mer DNA
primer annealed in such a way to a 55-mer DNA template that a
5
0 -overhang of 25 nucleotides was obtained (Fig. 4a, Table 1) and
compared this with a 80-mer/30-mer template/primer that comprised the same 5
0 -overhang as used in the SPR experiments of
Glover and McHenry [13].
To avoid degradation by contaminating nucleases, we used
oligonucleotides that contained four phosphorothioate (PTO)
bonds at both their 5
0 - and 3
0 - ends. A PTO bond is a phosphodiester bond where one nonbridging oxygen is replaced by sulfur
(Fig. 4c), and by this, it offers increased stability against nucleolytic
attacks. Template/primer constructs were obtained by annealing
the respective template with an excess of primer followed by SEC to
remove non-hybridized primer (Fig. 4b) and were checked for
homogeneity by AUC and gel electrophoresis (Fig. 4d, e).
1.5 Determination
of the Affinity of an
Interaction by
Sedimentation Velocity
Experiments
Whenever two molecules interact, the resulting complex will have a
higher molecular mass and will usually sediment faster than the two
components on their own. If the interaction is fast on the time scale
of sedimentation (dissociation rate constant >10
À3 s
À1 [19]), the
reaction will continue during the sedimentation process, which
typically takes several hours. The complex will dissociate and reassociate during sedimentation and will therefore not sediment as a
single species in an extra boundary, but together with one of the
interaction partners in a so-called reaction boundary. Whether this
is the slower (A) or the faster (B) sedimenting interaction partner
depends on the composition of the reaction mixture. If an excess of
A is present, it can be assumed as a good approximation that the
Analysis of Protein-DNA Interactions by AUC
403
proteins containing an LPXTG recognition motif to the amino
group of a pentaglycine on the peptidoglycan cell wall [17]. This
enzyme can be used in vitro to covalently link a labeled peptide
containing an N-terminal glycine residue to a protein with a SrtA
recognition site (Fig. 3a). As the MBP is positioned at the
C-terminus of the recognition site, it is cleaved off during the
labeling reaction and it can easily be judged by SDS-PAGE whether
the labeling is completed (Fig. 3c). Following the labeling reaction,
the FITC-labeled clamp loader was isolated by SEC (Fig. 3b, c) and
checked for homogeneity by AUC (Fig. 2).
1.4 Template/
Primer DNA
The sequences of the oligonucleotides were chosen according to
Bloom et al. [18], though omitting the 3
0 -overhang of the template
makes them thereby better comparable to the 105-mer/50 -fluorescinated 50-mer template/primer used in SPR [13]. However, they were designed to have shorter double-stranded regions
since only about ten nucleotides of double-stranded DNA have
been shown to be involved in interactions with the clamp loader
[15]. We used a template/primer consisting of a 30-mer DNA
primer annealed in such a way to a 55-mer DNA template that a
5
0 -overhang of 25 nucleotides was obtained (Fig. 4a, Table 1) and
compared this with a 80-mer/30-mer template/primer that comprised the same 5
0 -overhang as used in the SPR experiments of
Glover and McHenry [13].
To avoid degradation by contaminating nucleases, we used
oligonucleotides that contained four phosphorothioate (PTO)
bonds at both their 5
0 - and 3
0 - ends. A PTO bond is a phosphodiester bond where one nonbridging oxygen is replaced by sulfur
(Fig. 4c), and by this, it offers increased stability against nucleolytic
attacks. Template/primer constructs were obtained by annealing
the respective template with an excess of primer followed by SEC to
remove non-hybridized primer (Fig. 4b) and were checked for
homogeneity by AUC and gel electrophoresis (Fig. 4d, e).
1.5 Determination
of the Affinity of an
Interaction by
Sedimentation Velocity
Experiments
Whenever two molecules interact, the resulting complex will have a
higher molecular mass and will usually sediment faster than the two
components on their own. If the interaction is fast on the time scale
of sedimentation (dissociation rate constant >10
À3 s
À1 [19]), the
reaction will continue during the sedimentation process, which
typically takes several hours. The complex will dissociate and reassociate during sedimentation and will therefore not sediment as a
single species in an extra boundary, but together with one of the
interaction partners in a so-called reaction boundary. Whether this
is the slower (A) or the faster (B) sedimenting interaction partner
depends on the composition of the reaction mixture. If an excess of
A is present, it can be assumed as a good approximation that the
Analysis of Protein-DNA Interactions by AUC
403