fluorescence 3.2

Question 1

How many strands in GFP?

Answer 1

11

Question 2

Which organism was GFP isolated from?

Answer 2

Jellyfish Aequorea victoria

Question 3

How does GFP fluoresce in nature without UV/blue light excitations?

Answer 3

Protein Aequorin + Ca2+ + Co-factor –> Blue Light –> Activate GFP –> Green Light

Question 4

What is the function of GFP in Aequorea victoria?

Answer 4

Attract Mates, Seeing Food & Giving Alarming Signals for Defense

Question 5

What are some characteristics of wild type GFP?

Answer 5

-Small protein,
-Highly stable
-No physiological function in mammalian cells (No Gain-
of-Function)
-GFP-fusion proteins à Express in living cells
-Ex by UV

Question 6

How big is GFP?

Answer 6

27 kD, 238 aa residues

Question 7

How stable is GFP?

Answer 7

fluorescent up to 65o C, pH 11, even after fixation

Question 8

What ex and em wavelength of GFP?

Answer 8

ex by 470nm; emits fluoresces at 509 nm

Question 9

What is the definition of a chromophore?

Answer 9

A chemical group that absorbs light at a specific lamda for Ex & Em

Question 10

What are some limitations of wild type GFP?

Answer 10

• Slow maturation rate –> Oxidation of Fluorophore: 2-4
  hrs
• UV Excitation Probe
• Poor Folding at 37C
• Poor Photo-stability
• Dimerization –> Difficult to form GFP-fusion proteins
• pH Sensitive: Low pH –> No Em

Question 11

How does wtGFP mature?

Answer 11

Oxidation and Autoacylation (ser 65 to gly 67)
–> 2 steps

Question 12

What are the positions of the chromophore in wtGFP before maturation?

Answer 12

Ser 65
Tyr 66
Gly 67

Question 13

How were GFP variants produced?

Answer 13

Random Mutagenesis

Low-grade Taq Polymerase (Error-prone PCR) –> GFP Plasmids –> bacterial transformation –> DNA isolation and sequencing –> GFP variants with diff colors

Question 14

What is the mutation in enhanced GFP?

Answer 14

Serine –> Threonine at Position 65

Question 15

What were the improvements observed in enhanced GFP as compared with wtGFP?

Answer 15

1. Increased fluorescence intensity
2. can be Excited by Visible Light (e.g. 488 nm)
3. Increase in Stability & Brightness
4. No Dimerization
5. Maturation Rate: only 27 mins

Question 16

What could GFP variants used to study?

Answer 16

1. Luminous Tags for Tunelling Nanotube (TNT)
   2.

Question 17

What is the function of tunelling nanotubes between cells?

Answer 17

Cancer Cells Get Heathy Mitos from T Cells to Make ATP for Survival and Cancer Cells Send Damaged Mitos to T Cells to Block the T Cells’ Anti-cancer Activity.

Question 18

What is the full form of FRET?

Answer 18

Fluorescence Resonance Energy Transfer

Question 19

Explain the concept of FRET in simple terms

Answer 19

1. fluorochromes pair (donor & acceptor pair) –>
   emission of 1 overlap with excitation of the other
   (spectral overlapping)
2. when distance between 2 less than 10nm (i.e.
   receptor binding ) —-> FRET

Question 20

What are some applications of FRET?

Answer 20

1) PA - P B Interaction (Colocalization)
2) Reporter for Cell Activity

Question 21

What is FRET efficiency?

Answer 21

less than 100%

Question 22

Name the FRET censor used for Ca2+ sensing

Answer 22

Cameleon

Question 23

What does Cameleon consist of?

Answer 23

Calmodulin carrying CFP and M13 peptide carrying YFP.
Calmodulin and M13 linkedin by peptide linker

donor-acceptor: CFP-YFP

Question 24

State the steps involved in Ca2+ sensing using Cameleon

Answer 24

1. agnonist bind to receptor –> increase in Ca2+
2. Ca2+ bind to Calmodulin
3. changes in conformation of calmodulin
4. M13 pepride bind to Calmodulin
5. FRET

Question 25

What ex and em in Cameleon?

Answer 25

ex 440 em 530

Question 26

What is the donor and acceptor in Cameleon used for Ca2+ sensing?

Answer 26

donor: CFP (ex 440nm & ex 480nm) acceptor: YFP (ex 480nm & ex 530nm) Thus overall ex 440nm and em 530nm

Question 27

What are calmodulin and M13 linked by?

Answer 27

a peptide linker which provides flexibility but they bind to each other only when the conformation of Calmodulin changes after binding with Ca2+

Question 28

How many Ca2+ can bind to one Calmodulin?

Answer 28

4

Question 29

Describe how ratiometric analysis could be done using Cameleon

Answer 29

YFP emission at 530 nm (acceptor fluorescence, increases with calcium). CFP emission at 480 nm (donor fluorescence, decreases with calcium) Thus, 530/480

Question 30

State the +ve control when doing experiments using Cameleon

Answer 30

Ionomycin An Artificial Ca2+ Channel on Plasma Membrane --> max fluorescence (Fmax)

Question 31

What are the advantages of Cameleon?

Answer 31

1. non invaseive 2. cells produce indicator 3. binding to Ca2+ is reversible 4. 1 ex and 2 em lamda (Fura-2 --> 2 ex and 2 em) refer to notes for more pls