3
$\begingroup$

GFP ( green fluorescent protein) can show green fluorescence. And its fluorescence is due to the tri peptide chromophore which is given in below imageI was wondering, can we observe fluorescence, if we denature it by boiling at 95 degree celsius? Say can we observe fluorescence band in SDS-PAGE?

I assume it can show some fluorescence as it is not due to any cofactors etc.

$\endgroup$
2
$\begingroup$

According to PDB (protein data bank — a repository for protein structures) the chromophore must be protected from interaction with water molecules to fluoresce.

The chromophore is found right in the middle of the [protein], totally shielded from the surrounding environment. This shielding is essential for the fluorescence. The jostling water molecules would normally rob the chromophore of its energy once it absorbs a photon. But inside the protein, it is protected, releasing the energy instead as a slightly less energetic photon of light.

This means that denaturing the protein will result in quenching of the fluorescence1, so you are unlikely to see significant signal on a denaturing gel. In fact, as WYSIWYG has pointed out, fluorescence has been used as a marker of renaturation of GFP1.

Reference:

1: R.Y. Tsien (1998) The green fluorescent protein. Annual Review of Biochemistry 67, 509-544.

$\endgroup$
4
  • 2
    $\begingroup$ Additionally, there are biophysical studies on gfp folding in which increased fluorescence corresponds to folded state. $\endgroup$
    – WYSIWYG
    Jul 24 '19 at 7:50
  • $\begingroup$ @WYSIWYG any reference if you give it would be appreciated $\endgroup$
    – Science123
    Jul 25 '19 at 1:04
  • $\begingroup$ I wonder what would happen if you exchange the water in the gel with methanol or ethanol. Will these quench the fluorescence? $\endgroup$
    – BPinto
    Jul 25 '19 at 1:20
  • $\begingroup$ @Science123 check this out: pnas.org/content/109/44/17832 $\endgroup$
    – WYSIWYG
    Jul 26 '19 at 11:41

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.