[Protein-analysis] Re: Proteins Digest, Vol 29, Issue 1

[Sai Praveen]

Hi,
   
  I am planning to perform site-directed mutagenesis using PCR megaprimer  (~60bp). I was quite attracted by the Stratagene Quik-Change site-directed mutagenesis kit's simplicity. Please, if you had a chance to work with this kit, let me know if you had trouble with primer forming secondary structures.

  Thanks!
  Sai
  
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Today's Topics:

1. Drug binding: When fluorescence polarization fails
(Dominic-Luc Webb)
2. Re: Drug binding: When fluorescence polarization fails (Marvin)
3. Re: Drug binding: When fluorescence polarization fails
(Dominic-Luc Webb)
4. Course on Biomolecular Modelling (Patrick Sticher)


----------------------------------------------------------------------

Message: 1
Date: Mon, 1 Oct 2007 16:05:01 +0200
From: Dominic-Luc Webb 
Subject: [Protein-analysis] Drug binding: When fluorescence
polarization fails
To: proteins from net.bio.net
Message-ID: 

Content-Type: TEXT/PLAIN; charset=US-ASCII


I have worked for some time with fluroescence polarization (FP)
in studies of drug-protein binding. My drug, which is
hydrophobic, typically has a fluorescent adduct and the
solution has 0.01% Tween 20 to keep down non-specific binding
to test tubes, multiwell plates, etc. Assay usually works well.

I usually do a check with a series of glycerol concentrations
to ensure that FP is working OK. I expect at high glycerol
concentration, the higher viscosity lowers molecular rotation
which is detected as an increase in the amount of polarized
light in the static (parallel) channel. This usually works
fine, fluorescein for instance. I recently have found some
labelled drugs that show no increase in polarization, and
even a small decrease (about 20 mP) even when adding glycerol
to 50%. Again, Tween 20 is 0.01% throughout. Fluorescence
intensity remains relatively constant with only a slight
parallel increase in both channels. The underlying physics of
FP always seemed pretty straightforward, but now I am
confronted with such data that has been reproduced several
times and it is not clear how this came about.

Strangely, when a protein that is known to be a drug target is
present, there is a strong increase in polarization (100-300 mP).
The glycerol data has created an uncertainty about negative
data for test proteins that I am screening for drug binding.

Is there anyone in here that might have some hints how this
lack of change in polarization in glycerol might have come
about, maybe some suggestions how to troubleshoot this?

Dominic



------------------------------

Message: 2
Date: Mon, 01 Oct 2007 16:17:00 GMT
From: Marvin 

Subject: [Protein-analysis] Re: Drug binding: When fluorescence
polarization fails
To: proteins from net.bio.net
Message-ID: <049Mi.11183$bV2.6674 from trndny02>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Dominic-Luc Webb wrote:
> I have worked for some time with fluroescence polarization (FP)
> in studies of drug-protein binding. My drug, which is
> hydrophobic, typically has a fluorescent adduct and the
> solution has 0.01% Tween 20 to keep down non-specific binding
> to test tubes, multiwell plates, etc. Assay usually works well.
> 
> I usually do a check with a series of glycerol concentrations
> to ensure that FP is working OK. I expect at high glycerol
> concentration, the higher viscosity lowers molecular rotation
> which is detected as an increase in the amount of polarized
> light in the static (parallel) channel. This usually works
> fine, fluorescein for instance. I recently have found some
> labelled drugs that show no increase in polarization, and
> even a small decrease (about 20 mP) even when adding glycerol
> to 50%. Again, Tween 20 is 0.01% throughout. Fluorescence
> intensity remains relatively constant with only a slight
> parallel increase in both channels. The underlying physics of
> FP always seemed pretty straightforward, but now I am
> confronted with such data that has been reproduced several
> times and it is not clear how this came about.
> 
> Strangely, when a protein that is known to be a drug target is
> present, there is a strong increase in polarization (100-300 mP).

That is what I'd expect. Binding to a large molecule (i.e., 
the protein) reduces the rotation rate of the labeled drug, 
thus increasing the polarization.

> The glycerol data has created an uncertainty about negative
> data for test proteins that I am screening for drug binding.
> 
> Is there anyone in here that might have some hints how this
> lack of change in polarization in glycerol might have come
> about, maybe some suggestions how to troubleshoot this?

It could be that the change of solvent to 50% glycerol 
reduces the fluorescence lifetime, thus increasing the 
measured polarization.

> 
> Dominic
> 


------------------------------

Message: 3
Date: Tue, 2 Oct 2007 11:11:06 +0200
From: Dominic-Luc Webb 
Subject: [Protein-analysis] Re: Drug binding: When fluorescence
polarization fails
To: proteins from net.bio.net
Message-ID: 

Content-Type: TEXT/PLAIN; charset=US-ASCII


Thanks Marvin for your response, and sorry about any confusion I
might have created in the use of the word "strangely". The
"strangeness" had only to do with the disjunct between the
unexpected glycerol data and the expected protein binding data
using the same labelled drug. Until now, I regarded both of
these as positive control tests.

Regarding 50% glycerol, I could be convinced that the fluorescence
lifetime was changed, but I would need to bring this in line with
recent experiments. I went back and made some further measurements
and quantifications together with data I collected earlier. I
calculate a 1.4 fold increase in fluorescence intensity (FI) when
measured with normal FI filter set. Using the FP filters to measure
same samples, there was also an increase in fluorescence intensity in
both of the two channels (1.35 in the static channel, 1.41 in the
perpendicular channel). Fluorescence yield must have increased
concommitantly with a differential shift towards the perpendicular
channel.

Considering that last sentence, this assay has thus far not been
very sensitive to different intensities (e.g., fluorescein over a
few orders of magnitude). However, the differential shift towards
the perpendicular channel (1.41/1.35), although small, approaches
significance and seems in line with your suggestion that fluorescence
lifetime was decreased. Does this make sense?

Dominic


> > Strangely, when a protein that is known to be a drug target is
> > present, there is a strong increase in polarization (100-300 mP).
>
> That is what I'd expect. Binding to a large molecule (i.e.,
> the protein) reduces the rotation rate of the labeled drug,
> thus increasing the polarization.
>
> > The glycerol data has created an uncertainty about negative
> > data for test proteins that I am screening for drug binding.
> >
> > Is there anyone in here that might have some hints how this
> > lack of change in polarization in glycerol might have come
> > about, maybe some suggestions how to troubleshoot this?
>
> It could be that the change of solvent to 50% glycerol
> reduces the fluorescence lifetime, thus increasing the
> measured polarization.
>
> >
> > Dominic
> >
>



------------------------------

Message: 4
Date: Tue, 02 Oct 2007 11:21:58 +0200
From: Patrick Sticher 
Subject: [Protein-analysis] Course on Biomolecular Modelling
To: proteins from magpie.bio.indiana.edu
Message-ID: <47020DB6.5020504 from bioc.unizh.ch>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Dear colleagues,

please be informed that applications for the

6th NCCR PRACTICAL COURSE ON BIOMOLECULAR MODELLING

will be accepted still until October 10, 2007.

The course will take place January 6 - 11, 2008 as a winter retreat in 
the Swiss Alps, and will cover key topics in the area of computational 
structural biology. The course format includes morning lectures by 
experts in the field that alternate with later afternoon to evening 
tutorials and discussions.

For further details about this course, please visit the course website 
http://www.structuralbiology.unizh.ch/course2008.asp

If you are interested in this course, please use the online form on the 
course website to apply.

Best regards,
Patrick Sticher

-- 
_________________________________
Visit the NCCR on the Internet
www.structuralbiology.uzh.ch

Dr. Patrick Sticher Moser
NCCR Scientific Officer
Institute of Biochemistry
University of Zürich
Winterthurerstrasse 190
CH - 8057 Zürich

Phone +41 / (0)44 / 635 54 84
Fax +41 / (0)44 / 635 59 08
Mail sticher from bioc.uzh.ch



------------------------------

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