Kap 11/13: microscopy & spectroscopic techniques

Question 1

Molecular (sub)structures responsible for interaction with electromagnetic radiation
are called

Answer 1

chromophores

Question 2

Which three tpes of chromophoes are relevant if UV/Vis spectroscopy

Answer 2

peptide bonds (amide bonds - far UV)
certain Amino acid side chians (Tryptophan and tyrosine)
certain prosthetic groups and coenzymes

Question 3

The electronic transitions of the peptide bond occur in the far UV, which three peaks are relavant transitions

what about for amino acid side chains?

and for prosthetic groups?

Answer 3

190
210-220

> 230

420 , 450 and 480

Question 4

The chance of a photon being absorbed by matter is given by an:

Answer 4

extinction coeffi cient ,

Question 5

The enviroment of a chromophoes also affects the observed spectrum, which can be described by three parameters, which three?

Answer 5

• protonation/deprotonation (pH, redox)
• solvent polarity (dielectric constant of the solvent)
• orientation effects.

Question 6

oxidation/reduction reactions –> solvent polarity changes –> 4 effects

Answer 6

blue shift/red shift

hypo/hyperchromicity

Question 7

What gives rise to the CD phenomenon of

a protein in the wavelength interval 190–260 nm.

Answer 7

the peptide bond; amide bond with the vicinal carbon atom that is asymmetric and a stereogenic center in all amino acids except glycine –> this chirality inudces asymmetry

Question 8

Proteins possess two intrinsic fluorophores: :

Answer 8

tryptophan and tyrosine

Question 9

The main application for intrinsic protein fl uorescence aims at:

Answer 9

conformational monitoring

Question 10

A number of empirical rules can be applied to interpret protein fluorescence spectra:

1. As a fluorophore moves into an environment with less polarity, its emission spectrum exhibits a ____ shift (moves to shorter wavelengths), and the intensity
   at increases.

Answer 10

hypsochromic

Question 11

Fluorophores in a polar environment show a decrease in ___ ___ with increasing
temperature. In a non-polar environment, there is little change.

Answer 11

quantum yield

Question 12

The excitation and emission wavelengths of this photo-activatable __ are
essentially identical to those of native GFP ( wavelength exc = 488 nm, wavelength em = 509 nm);
however,
the fluorescence emission intensity is increased 100-fold after an ‘activating’ light
pulse at 413 nm.

Answer 12

GFP

Question 13

What is a quencher?

Answer 13

a molcules that asorbs energy from an excited state via a non-radiativ process thus decreasing quantum yeild

Question 14

Fluorescence Polarisation

1. Absorption of polarised light by a chromophore is highest when the plane of polarisation is parallel to the:
2. Fluorescence emission, in contrast, does not depend on the absorption dipole moment, but on the:
3. usually these two moments are tilted against each other about:

Answer 14

absorption dipole moment
transition dipole moment
10* to 40*