Protein photoswitches... photo-isomerizable chemical structures

[Nathan McCorkle]

So it seems adding a photoswitch isn't terribly hard, but it's an in-vitro operation as far as I've been reading... at least we haven't figured out the synBio way to do it yet.

This is the seemingly simplest photo-isomerizable group:

http://en.wikipedia.org/wiki/Azobenzene

goes from trans to cis when you illuminate it, so if you add this between areas of a protein in the right way, you can twist the protein into or out of a working conformation via a pulse of light. 

The way to engineer a protein to receive the current in-vitro treatment is to identify all the surface exposed amino acids, find two that are about the distance relaxed or excited photoswitch, then try to engineer them to be cysteines and try to engineer away any other surface cysteines. Then express the engineered protein with your specially placed cysteine pair, purify the protein, add maleimide linker by using this cross-linker

http://en.wikipedia.org/wiki/Maleimide

then hope that it works, and if not try again. Here's a good slideshow of the concept:

Molecular Photoswitches – Properties and Applications 

Shishi Lin, Organic Student Seminar, Yoon Group

http://www.chem.wisc.edu/areas/organic/studsemin/lin_shishi/lin-sem.pdf

It looks like we're getting close to understanding the chemistry of natural systems a bit better...

Reversible Photocontrol of Peptide Conformation with a Rhodopsin-like Photoswitch

http://pubs.acs.org/doi/abs/10.1021/ja301868p

Sooo, what are some existing biological light switches that people have seen hacked or are ripe to be?

I'm wondering if one could make a channel that spits out single nucleotides when illuminated, or maybe use a neuron that blebs out a vesicle with just one nucleotide in it. (in effort of directed DNA synthesis)

--
-Nathan

[Sebastian S. Cocioba]

If you look into optogenetics, the channel rhodopsin pathway is very well understood and is being used in triggering cardio myocytes, neurons, etc with a pulse of light. The channel rhodopsin integration vector, tied to CMV, is used to transform mammalian cells but the actual ion channel came from "Clamy" algae cells. It has very high genetic similarity to the g protein linked receptors in the retina and works very well with few photons needed.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diy...@googlegroups.com.
To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio?hl=en.
For more options, visit https://groups.google.com/groups/opt_out.
 
 

[Nathan McCorkle]

Do you know of any work in prokaryotes?

--
-Nathan

[Nathan McCorkle]

Ahh here is one where they're simply making a fusion protein! It's
open-access too!

LOVely enzymes – towards engineering light-controllable biocatalysts
Ulrich Krauss, Jeeyeon Lee, Stephen J. Benkovic, Karl-Erich Jaeger
http://onlinelibrary.wiley.com/doi/10.1111/j.1751-7915.2009.00140.x/abstract

--
-Nathan

[Bryan Bishop]

On Sat, Jan 5, 2013 at 12:51 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
> LOVely enzymes – towards engineering light-controllable biocatalysts
> Ulrich Krauss, Jeeyeon Lee, Stephen J. Benkovic, Karl-Erich Jaeger

I figure Anselm will eventually weigh in..

http://anselmlevskaya.com/publications/Nature%202009%20Levskaya.pdf

- Bryan
http://heybryan.org/
1 512 203 0507

[Patrik D'haeseleer]

A few more from Anselm:

Engineering Escherichia coli to see light

Multichromatic Control of Gene Expression in Escherichia coli

Patrik

[Josiah Zayner]

There is a lot of working currently being done in this field outside of channels.

The main competitors are:
LOV domains
Phytochromes
Cryptochromes

Currently phytochromes and cryptochromes seem to have to best light/dark switching but they come at the cost of the proteins being huge(> 100kDa) and the need to add exogenous chromophore (billins).

Personally I work on LOV domains which are small(<15kDa) and contain an FMN chromophore found in most every cell but we do not quite understand how they work and how to make the conformational switching better. We are becoming more knowledgeable though. However, LOV domains have been used to do cool stuff like make cells move, and colocalize substrates.

My Ph.D. thesis is on characterizing the biophysical phenomena regarding LOV domains and apply that to make good photoswitchable proteins. Currently anyone can make two proteins colocalize or turn on a gene. I think what everyone wants or is trying to do is to make most "any" protein function activatable by light. Something cryptochromes and phytochromes will not be able to do this easily because of their size and need to for exogenous chromophore. However, even in the context of LOV domains making something like the well characterized KcsA potassium channel light activated is no easy task.


I can easily provide plasmids for engineered LOV domains if requested.

[Nathan McCorkle]

Whoo! Thanks Josiah,  it may be a year or so until I'd want plasmids,  hopefully you'll have something in the freezer then!

--

-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diy...@googlegroups.com.
To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio?hl=en.

To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/6rc22zBMvJgJ.

[Ulysses1994XF04]

Are you aware of any online resources (databases, whatever) that have the 3D structures of enzymes calculated and mapped?

[Eugen Leitl]

On Sat, Jan 05, 2013 at 09:28:45PM -0800, Ulysses1994XF04 wrote:
> Are you aware of any online resources (databases, whatever) that have the
> 3D structures of enzymes calculated and mapped?

Of course: http://en.wikipedia.org/wiki/Protein_Data_Bank