14 | Basic Techniques Probing Molecular Structure and Interactions

Question 1

What label-free technique probes the thermodynamics of a system under a binding event in solution ?
Explain the method?

Answer 1

1. ITC
2. It quantifies small heat changes to a cell by detecting energy required to maintain the cell at constant temperature, as a series of spikes over time, each spike is then integrated and summed giving total heat exchange per injection. A plot is then made of enthalpy change against the molar ratio of the ligand to enzyme.

Question 2

ITC has been successfully used for determination of ?

Answer 2

rates of enzymatic reactions
and their use in investigation of enzyme
kinetics.

Question 3

A further important application of ITC has been in the study of the interconversion of ___ _____ and the elucidation of ____ of ____

Answer 3

protein conformations
mechanism of allostery

Question 4

An incremental increase of a component or an inhibitor addition in ITC allows the study of?

Answer 4

reaction mechanisms

Question 5

____-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.

Answer 5

Switch

Question 6

The analysis of molecular interactions can result in the development of a biosensor, explain this devise?

Answer 6

Is composed of a biological element and a physio-chemical transduction part that convert signal reception by the biological entity into a physical quantifiable response

Question 7

Explain FRET!

Answer 7

A switch technique where changes in molecular states associated with the pairing of two molecules is adapted into a assay to analyse inter-molecular interactins. where a fluorescent molecule (the donor) has its excitation light absorbed if the acceptor molecule is in close proximity

Question 8

Explain SPR!

Answer 8

TIR of an incident light beam at the prism–metal interface elicits a propagating plasmon wave in par as a longitudual vibration in the conducting band of the metal due to vibrational excitation at a resonance frequency – leading to an electrical field intensity, called an evanescent field wave, that leaks into the medium of lower refractive index where it decays at an exponential rate and effectively only travels one wavelength – giving in hindsight a decreased intensity of the reflected light at a specific angle of reflection because the plasmons transmuted the energy of the incident photons.
When binding accurs there’s a mass change, increasing the refractive index thereby altering the angle of reflected light – eliciting a change in the detector position, which can be plotted against time to give a sensorgramme reading on whether a binding has occurred.
An array detector allows the creation of an image that give better lateral resolution.