Column 7: First binding partner P 1 at the same fixed concentration and varying concentrations of the second partner
P 2, ideally in the range 0.1 Â K d to 10 Â K d for P 2 binding
to N, if known. This concentration range might need to be
changed if P 1 and P 2 bind with very different affinities.
3. Program the experiment to perform the following steps:
Steps (a)–(c) Baseline 1, Loading, Baseline 2: As in step 5 in
Subheading 3.1.
Step (d) Association 1: The sensors are dipped in column
5, containing P 1 . In this step the binary complex P 1 N is
formed.
Step (e) Association 2: The sensors are dipped in column
7, containing P 1 and P 2 . In this step, some of P1 will
dissociate and some of P2 will bind.
Step (f) Dissociation 1: The sensors are dipped in column
6, containing only P 1 . In this step, the P2 should dissociate
and P1 should rebind.
Step (g) Dissociation 2 (optional): The sensors are dipped in
buffer column 3, to measure the dissociation of P 1 .
3.4 Data Analysis
In recent years, we have employed the analytical approach described
in Subheading 1.3 to determine kinetic (k on and k off ) and thermodynamic (K d ) parameters for the interaction of different proteins
with their DNA and RNA target sequences [11–16] and also
showed that it is possible, starting from those data, to build a
kinetic model for the interaction that provides information on
mRNA regulation and RNA remodeling [15, 16].
IMP1/ZBP1 is a multifunctional RNA-binding protein that
regulates mRNA metabolism, transport, and translation during
development and in cancer [17]. It contains six putative
RNA-binding domains (two RRM and four KH) organized in
three two-domain units. Interestingly, the binding of different
RNA targets is mediated by the two KH di-domains, KH1KH2
and KH3KH4, in a target-dependent fashion. For example, binding of the c-Myc oncogene mRNA in highly proliferating cells
requires the KH1KH2 di-domain [18], while the interaction of
IMP1 with the β-actin mRNA in neurons requires only the
KH3KH4 di-domain [19].
In a recent study, we examined the interaction of KH1KH2
with an oligo recapitulating an IMP1-binding site (CACAGCAUA
CAUCCUGUCCGUC ), which we named MYCRNA [16]. An
important tool to dissect this interaction has been a KH domain
mutant where nucleic acid binding is eliminated by the mutation of
the two variable amino acids in the hallmark GxxG loop to Aspartate (GxxG-to-GDDG) [12, 13]. This mutation does not affect the
structure or the stability of the domain and allows one to examine
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Stephen R. Martin et al.
P 2, ideally in the range 0.1 Â K d to 10 Â K d for P 2 binding
to N, if known. This concentration range might need to be
changed if P 1 and P 2 bind with very different affinities.
3. Program the experiment to perform the following steps:
Steps (a)–(c) Baseline 1, Loading, Baseline 2: As in step 5 in
Subheading 3.1.
Step (d) Association 1: The sensors are dipped in column
5, containing P 1 . In this step the binary complex P 1 N is
formed.
Step (e) Association 2: The sensors are dipped in column
7, containing P 1 and P 2 . In this step, some of P1 will
dissociate and some of P2 will bind.
Step (f) Dissociation 1: The sensors are dipped in column
6, containing only P 1 . In this step, the P2 should dissociate
and P1 should rebind.
Step (g) Dissociation 2 (optional): The sensors are dipped in
buffer column 3, to measure the dissociation of P 1 .
3.4 Data Analysis
In recent years, we have employed the analytical approach described
in Subheading 1.3 to determine kinetic (k on and k off ) and thermodynamic (K d ) parameters for the interaction of different proteins
with their DNA and RNA target sequences [11–16] and also
showed that it is possible, starting from those data, to build a
kinetic model for the interaction that provides information on
mRNA regulation and RNA remodeling [15, 16].
IMP1/ZBP1 is a multifunctional RNA-binding protein that
regulates mRNA metabolism, transport, and translation during
development and in cancer [17]. It contains six putative
RNA-binding domains (two RRM and four KH) organized in
three two-domain units. Interestingly, the binding of different
RNA targets is mediated by the two KH di-domains, KH1KH2
and KH3KH4, in a target-dependent fashion. For example, binding of the c-Myc oncogene mRNA in highly proliferating cells
requires the KH1KH2 di-domain [18], while the interaction of
IMP1 with the β-actin mRNA in neurons requires only the
KH3KH4 di-domain [19].
In a recent study, we examined the interaction of KH1KH2
with an oligo recapitulating an IMP1-binding site (CACAGCAUA
CAUCCUGUCCGUC ), which we named MYCRNA [16]. An
important tool to dissect this interaction has been a KH domain
mutant where nucleic acid binding is eliminated by the mutation of
the two variable amino acids in the hallmark GxxG loop to Aspartate (GxxG-to-GDDG) [12, 13]. This mutation does not affect the
structure or the stability of the domain and allows one to examine
360
Stephen R. Martin et al.