Chapter 14: Basic Techniques Probing Molecular Structure and Interactions Flashcards
ITC is a technique probing what?
is it considered an in-direct technique?
the thermodynamics of a system of binding partners in solution
no
What does ITC detect and quantifies?
small heat changes associated with the binding
What advantage does ITC have in regard to reacting species?
they dont need to be chemically modified or immobilised.
ITC:
enthalpy change vs. ______
molar ratio of the ligand to enzyme
ITC
what type is the plot
an from the plot what can be calulated?
hyperbolic
enthalpy, free energy
and entropy changes associated with the ligand binding and hence the dissociation
constant, Kd , and stoichiometry of binding n
What does ITC investigate?
thermodynamic parameters and kinetic information on molecular interaction processes in solution
ITC has been successfully used for:
and what other important application of ITC has been in the study of?
determination of rates of enzymatic reactions and their use in investigation of enzyme kinetics
interconversion of protein conformations and the elucidation of the mechanism
of allostery
In ITC if we are to incremental increase of a component or an inhibitor, what does this allow us to study?
reaction mechanisms
If we combine ITC with chromatography what can this be used for?
to identify a target protein for a particular
ligand within a biomolecular mixture.
What is elstic light scattering aslo known to be called?
Rayleigh scattering
What type of techniques can be perfomed in solution with minimal procedual steps?
switch
Switch-like behaviour can be enacted through changes in:
molecular states associated
with the pairing of two molecules. This switch-like behaviour can be adapted
into assays that enables analysis of either inter-molecular or intra-molecular
interactions.
What is the main principle behind Switch techniques?
in which ways can it be used?
The distance between molecules can be assessed by labelling with pairs of molecules that produce a change in molecular state when they are brought into close proximity
- conform two parts within a molecule interact by a conf. change (intra-molecular analysis)
2.effects of potential inhibitors or enhancers - measure the affinity of that interaction by
measuring the amount of change or signalling that occurs (inter-molecular analysis) between two interacting molecules
Regarding FRET if the acceptor molecule is in close proximity to donor what happens?
the fluorescent molecule (the donor) has its
excitation light absorbed
since the basis of the mechanism for FRET functions as a molecular analysis tool, so regarding measurments what does it serve as?
a molecular ruler to determine the distance between two biomolecules
FRET
so regarding measurments it serves as a molecular ruler to determine the distance between two biomolecules, give one exampe how it can be applied!
For example, the molecular beacon
(see below) is used to measure the hybridisation of DNA molecules, and is emerging as
a biosensing tool for real-time measurement of analyte
FRET has also been applied to
study the, name several applications!
structure and conformation of proteins, the spatial distribution and assembly of proteins, receptor/ligand interactions, immune assays, structure and confirmation of nucleic acids, real-time PCR assays and single nucleotide polymorphism (SNP) detection, nucleic acid hybridisation events, distribution and transport of lipids, membrane fusion assays, membrane potential sensing and fluorogenic protease assays
FRET
what is the name of the probes used?
what are they designed for?
What happens when the probe is in the prescence of the target nucleic acid?
molecular beacons
to bind specifically to a target nucleic acid
the stem region of the beacon becomes separated,
releasing the fl uorophore from the quencher such that active fl uorescence can be
observed
FRET
The molecular dynamics of hybridisation enables:…
enables near-instantaneous realtime
reporting on the presence or absence of the desired target
FRET
The reactions are also very specific, enabling discrimination between target sequences that differ by a ___ nucleotide substitution (e.g. for SNP detection).
single
Can FRET be used for real-time PCR quantification?
yes
Assays to determine binding between two ligands can be performed using several different formats, which 4?
direct
indirect
sandwhich
competetive
- 6 SOLID-PHASE BINDING TECHNIQUES COMBINED WITH FLOW
- 6.1 Surface Plasmon Resonance :)
What does this method monitor?
Replacing
the ligand solution by a buffer solution enables investigation of the dissociation of ____ ligand.
The change in angle brings about a change in detector position, which can be plotted against time to give a sensorgramme reading, which indicates?
the value of the refractive index determines: …
In general, the ____ the wavelength of the light
used, the better the sensitivity, but at lower lateral resolution
Generally, all two-component binding reactions can be investigated, which opens a variety of applications in the areas of drug design (protein–___ interactions), as
well as mechanisms of membrane-associated proteins (____–membrane binding) and DNA-binding proteins
small changes in the refractive index or the thickness of thin fi lms. It is mainly used for monitoring the interaction of two components (e.g. ligand and receptor), one of
which is immobilised on a sensor chip surface
bound
when ligand is bound and unbound at different resonance signals
the propagation velocity of electromagnetic radiation in that medium.
higher
ligand
protein
With which stability quantification method according to Katarina can ALPHA-Screen ® be used with in regards to analysis?
ALPHA screening has been demonstrated
to be useful in the analysis of: …
CETSA
affinity interactions of small binding proteins,
studies on phosphorylation and oligomerisation, and other enzymatic and binding reactions.
FACS
a fl uorescently labelled probe indicates the presence or absence of cells with a specific ____ of interest
This technique is highly useful in: …
biomarker
immunology,
where subpopulations of cells containing specifi c biomarkers can be captured and
sorted
Why is SPR used extensively used in proteomic research?
the kinetics of receptor–ligand, antibody–antigen and protein–protein interactions
