Chapter 14: Basic Techniques Probing Molecular Structure and Interactions

Question 1

ITC is a technique probing what?

is it considered an in-direct technique?

Answer 1

the thermodynamics of a system of binding partners in solution

no

Question 2

What does ITC detect and quantifies?

Answer 2

small heat changes associated with the binding

Question 3

What advantage does ITC have in regard to reacting species?

Answer 3

they dont need to be chemically modified or immobilised.

Question 4

ITC:

enthalpy change vs. ______

Answer 4

molar ratio of the ligand to enzyme

Question 5

ITC
what type is the plot

an from the plot what can be calulated?

Answer 5

hyperbolic

enthalpy, free energy
and entropy changes associated with the ligand binding and hence the dissociation
constant, Kd , and stoichiometry of binding n

Question 6

What does ITC investigate?

Answer 6

thermodynamic parameters and kinetic information on molecular interaction processes in solution

Question 7

ITC has been successfully used for:

and what other important application of ITC has been in the study of?

Answer 7

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

Question 8

In ITC if we are to incremental increase of a component or an inhibitor, what does this allow us to study?

Answer 8

reaction mechanisms

Question 9

If we combine ITC with chromatography what can this be used for?

Answer 9

to identify a target protein for a particular

ligand within a biomolecular mixture.

Question 10

What is elstic light scattering aslo known to be called?

Answer 10

Rayleigh scattering

Question 11

What type of techniques can be perfomed in solution with minimal procedual steps?

Answer 11

switch

Question 12

Switch-like behaviour can be enacted through changes in:

Answer 12

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.

Question 13

What is the main principle behind Switch techniques?

in which ways can it be used?

Answer 13

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

1. conform two parts within a molecule interact by a conf. change (intra-molecular analysis)
   2.effects of potential inhibitors or enhancers
2. measure the affinity of that interaction by
   measuring the amount of change or signalling that occurs (inter-molecular analysis) between two interacting molecules

Question 14

Regarding FRET if the acceptor molecule is in close proximity to donor what happens?

Answer 14

the fluorescent molecule (the donor) has its

excitation light absorbed

Question 15

since the basis of the mechanism for FRET functions as a molecular analysis tool, so regarding measurments what does it serve as?

Answer 15

a molecular ruler to determine the distance between two biomolecules

Question 16

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!

Answer 16

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

Question 17

FRET has also been applied to

study the, name several applications!

Answer 17

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

Question 18

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?

Answer 18

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

Question 19

FRET

The molecular dynamics of hybridisation enables:…

Answer 19

enables near-instantaneous realtime

reporting on the presence or absence of the desired target

Question 20

FRET
The reactions are also very specific, enabling discrimination between target sequences that differ by a ___ nucleotide substitution (e.g. for SNP detection).

Answer 20

single

Question 21

Can FRET be used for real-time PCR quantification?

Answer 21

yes

Question 22

Assays to determine binding between two ligands can be performed using several different formats, which 4?

Answer 22

direct
indirect
sandwhich
competetive

Question 23

14. 6 SOLID-PHASE BINDING TECHNIQUES COMBINED WITH FLOW
15. 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

Answer 23

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

Question 24

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: …

Answer 24

CETSA

affinity interactions of small binding proteins,
studies on phosphorylation and oligomerisation, and other enzymatic and binding reactions.

Question 25

FACS
a fl uorescently labelled probe indicates the presence or absence of cells with a specific ____ of interest

This technique is highly useful in: …

Answer 25

biomarker

immunology,
where subpopulations of cells containing specifi c biomarkers can be captured and
sorted

Question 26

Why is SPR used extensively used in proteomic research?

Answer 26

the kinetics of receptor–ligand, antibody–antigen and protein–protein interactions