calculate the free energy of transition to each state (values along the
arrows) and subsequent calculations of ΔΔG. The results of these
calculations indicate that the first change along either pathway has
significant thermodynamic worth, the second change has essentially
no thermodynamic worth, and the coupling free energy indicates
positive cooperativity. Thus, these data suggest that functional
groups A and B participate in the same hydrogen bonding, since
their individual mutations remove the hydrogen bond, and once
the bond is removed it can no longer be used by the second
functional group.
2.2 Methods
to Determine
Cooperativity
and Detect Allosteric
Modulations
Over 100 years of research has expanded methodology to study
cooperativity and allostery, from measuring hemoglobin saturation
with oxygen as a function of the partial pressure of oxygen to many
more approaches, including detailed structural studies. However,
spectroscopy techniques remain the oldest and most popular methods for determining nucleic acid thermodynamics because of simplicity and low cost. In RNA spectroscopy, the signal is
proportional to the advance of the reaction as the molecules
undergo structural transitions brought on by either ligand binding
or changes in temperature. Conformational rearrangements are
accompanied by changes in intrinsic UV absorbance, which depend
on the formation of base pairs [11]. Another method for spectroscopically detecting conformational changes is through fluorescence [12, 13], for example, by internally incorporating
fluorescent nucleotide analog 2-aminopurine [14]. As an RNA
molecule folds upon ligand binding, the attached chromophore is
exposed to a different microenvironment, which alters fluorescence
intensity and the emission spectrum. Fluorescent assays typically
have high sensitivity since the chromophore’s properties strongly
depend on the microenvironment. A more sophisticated spectroscopic method to study conformational transitions in RNA is
nuclear magnetic resonance (NMR) (reviewed in [15]). Since
NMR signals depend on the microenvironment of atoms, they
can provide information about protonation, interactions, and
local structure, and thus they can be effectively used to study a
complex behavior such as cooperativity [16] .
While spectroscopy is extremely useful, technological advances
over the last several decades have given rise to the use of isothermal
titration calorimetry (ITC) as a means to measure the heat associated with interactions between biological molecules [17]. In this
technique, the solution of a macromolecule is located inside the
sample cell directly adjacent to a reference cell and the ligand
solution in the injector syringe. The ligand solution is injected
periodically into the sample cell, and each injection triggers the
binding reaction and formation of the complex. As the sequence
of injections proceeds, the heat associated with each injection is
proportional to the increase in complex concentration.
RNA Cooperativity and Allostery
259
arrows) and subsequent calculations of ΔΔG. The results of these
calculations indicate that the first change along either pathway has
significant thermodynamic worth, the second change has essentially
no thermodynamic worth, and the coupling free energy indicates
positive cooperativity. Thus, these data suggest that functional
groups A and B participate in the same hydrogen bonding, since
their individual mutations remove the hydrogen bond, and once
the bond is removed it can no longer be used by the second
functional group.
2.2 Methods
to Determine
Cooperativity
and Detect Allosteric
Modulations
Over 100 years of research has expanded methodology to study
cooperativity and allostery, from measuring hemoglobin saturation
with oxygen as a function of the partial pressure of oxygen to many
more approaches, including detailed structural studies. However,
spectroscopy techniques remain the oldest and most popular methods for determining nucleic acid thermodynamics because of simplicity and low cost. In RNA spectroscopy, the signal is
proportional to the advance of the reaction as the molecules
undergo structural transitions brought on by either ligand binding
or changes in temperature. Conformational rearrangements are
accompanied by changes in intrinsic UV absorbance, which depend
on the formation of base pairs [11]. Another method for spectroscopically detecting conformational changes is through fluorescence [12, 13], for example, by internally incorporating
fluorescent nucleotide analog 2-aminopurine [14]. As an RNA
molecule folds upon ligand binding, the attached chromophore is
exposed to a different microenvironment, which alters fluorescence
intensity and the emission spectrum. Fluorescent assays typically
have high sensitivity since the chromophore’s properties strongly
depend on the microenvironment. A more sophisticated spectroscopic method to study conformational transitions in RNA is
nuclear magnetic resonance (NMR) (reviewed in [15]). Since
NMR signals depend on the microenvironment of atoms, they
can provide information about protonation, interactions, and
local structure, and thus they can be effectively used to study a
complex behavior such as cooperativity [16] .
While spectroscopy is extremely useful, technological advances
over the last several decades have given rise to the use of isothermal
titration calorimetry (ITC) as a means to measure the heat associated with interactions between biological molecules [17]. In this
technique, the solution of a macromolecule is located inside the
sample cell directly adjacent to a reference cell and the ligand
solution in the injector syringe. The ligand solution is injected
periodically into the sample cell, and each injection triggers the
binding reaction and formation of the complex. As the sequence
of injections proceeds, the heat associated with each injection is
proportional to the increase in complex concentration.
RNA Cooperativity and Allostery
259