Despite the large number of RNA-binding proteins, recognition of the RNA targets is mediated by a relatively small number of
different RNA-binding domains, which are present in multiple
copies of the same or different domains within one RNA-binding
protein [5]. In these proteins, RNA binding is mediated by a
combinatorial action of more than one RNA-binding domain. In
this context, diversity of recognition stems from both the specificity
of a domain in a given protein and from a range of different interdomain coupling modes. Inter-domain coupling can increase affinity and specificity, reshape the RNA structure, and provide new
opportunities for regulation [6].
While a global survey of RNA-binding domains shows different
sizes and RNA recognition properties, the most common of these
domains are less than 100 amino acids in size. For many of these
domains, a structural characterization of a “canonical” binding
mode is available, together with, in some cases, the description of
a few of the structural and RNA-binding variations on this mode
[5, 6]. However, information on the kinetics of binding and interdomain coupling is available only in a small number of systems. This
information is essential to model the binding of these domains in
the cellular environment.
As an example of the RNA-binding domains discussed above,
the KH domain is a small (~70 amino acids) αβ fold found in a
number of RNA regulatory proteins important in development,
function, and disease. The domain binds to single-stranded nucleic
acids with a varying degree of affinity and specificity. RNA binding
is mediated by the interaction of the nucleic acid backbone with a
negatively charged GxxG loop [7]. The details of this interaction
are different in different domains, but binding of the loop orients
the nucleobases towards a hydrophobic groove in the protein, for
sequence specific recognition [7]. In addition, individual KH
domains can interact using a variety of surfaces. Importantly,
despite the importance of KH-containing proteins in human
health, kinetic and mechanistic information on multi-domain binding has only recently started to become available. This is partly due
to the difficulties in obtaining high-quality data on the kinetics of
protein–RNA interactions at different affinities in the same experimental system.
1.2 Biolayer
Interferometry
Biolayer interferometry (BLI) is a label-free method that enables
real-time analysis of biomolecular interactions occurring on 8 or
16 biosensors in 96- or 384-well plates [8–10]. White light travels
down the biosensors and is reflected back to spectrometers from
two places: an internal reference layer and the interface between the
solvent and molecules immobilized on the sensor tip. This results in
an interference pattern, and the instrument measures the maximum
wavelength of the pattern.
352
Stephen R. Martin et al.
different RNA-binding domains, which are present in multiple
copies of the same or different domains within one RNA-binding
protein [5]. In these proteins, RNA binding is mediated by a
combinatorial action of more than one RNA-binding domain. In
this context, diversity of recognition stems from both the specificity
of a domain in a given protein and from a range of different interdomain coupling modes. Inter-domain coupling can increase affinity and specificity, reshape the RNA structure, and provide new
opportunities for regulation [6].
While a global survey of RNA-binding domains shows different
sizes and RNA recognition properties, the most common of these
domains are less than 100 amino acids in size. For many of these
domains, a structural characterization of a “canonical” binding
mode is available, together with, in some cases, the description of
a few of the structural and RNA-binding variations on this mode
[5, 6]. However, information on the kinetics of binding and interdomain coupling is available only in a small number of systems. This
information is essential to model the binding of these domains in
the cellular environment.
As an example of the RNA-binding domains discussed above,
the KH domain is a small (~70 amino acids) αβ fold found in a
number of RNA regulatory proteins important in development,
function, and disease. The domain binds to single-stranded nucleic
acids with a varying degree of affinity and specificity. RNA binding
is mediated by the interaction of the nucleic acid backbone with a
negatively charged GxxG loop [7]. The details of this interaction
are different in different domains, but binding of the loop orients
the nucleobases towards a hydrophobic groove in the protein, for
sequence specific recognition [7]. In addition, individual KH
domains can interact using a variety of surfaces. Importantly,
despite the importance of KH-containing proteins in human
health, kinetic and mechanistic information on multi-domain binding has only recently started to become available. This is partly due
to the difficulties in obtaining high-quality data on the kinetics of
protein–RNA interactions at different affinities in the same experimental system.
1.2 Biolayer
Interferometry
Biolayer interferometry (BLI) is a label-free method that enables
real-time analysis of biomolecular interactions occurring on 8 or
16 biosensors in 96- or 384-well plates [8–10]. White light travels
down the biosensors and is reflected back to spectrometers from
two places: an internal reference layer and the interface between the
solvent and molecules immobilized on the sensor tip. This results in
an interference pattern, and the instrument measures the maximum
wavelength of the pattern.
352
Stephen R. Martin et al.