Well, I just got word that the protein is ~100kD anyway, so I think
the HSQC is out the window anyway!
Jacob
On Mon, Dec 6, 2010 at 12:16 PM, Roopa Thapar <[log in to unmask]> wrote:
>
>
> I agree that the experiment is a good one and can easily be done, but without assignments I think the interpretation could be ambiguous.
>
> pH dependent chemical shift perturbations could occur far removed from the linker (either due to a conformational change or the change in chemical environment around the amide nucleus)
> and without any information about which residues are shifting it may be difficult to conclude that these perturbations are due to a change in domain orientation rather than other subtle
> pH dependent effects.
>
> If there are no perturbations, then of course one can conclude little or no conformational changes occur. The magnitude of the perturbation would depend on how extensive the conformational change is.
>
> One could specifically label the protein with 15N-labeled amino acids that are particularly unique to the linker - this would simplify the spectrum and the data may be easier to interpret.
>
> It is a good experiment to try however.
>
>
> Roopa
>
>
>
> ________________________________________
> From: Jacob Keller [[log in to unmask]]
> Sent: Monday, December 06, 2010 12:54 PM
> To: Roopa Thapar
> Cc: [log in to unmask]
> Subject: Re: [ccp4bb] pH dependent conformational change
>
> Even without assignments, wouldn't a dramatic shift be seen in the
> interacting residues? Also, I suggested the method because it is
> pretty easy, probably doable in a week...
>
> Jacob
>
> On Mon, Dec 6, 2010 at 11:24 AM, Roopa Thapar <[log in to unmask]> wrote:
>> If there are backbone NMR assignments available then, definately a pH titration using HSQCs would give site specific information. These are easy experiments if someone can help you set them up.
>> The perturbations should map to the inter-domain interface.
>>
>> If there are no assignments for the protein, spectral changes in response to pH would be harder to interpret. You could try FRET by introducing two probes - one in each domain.
>>
>> Roopa
>>
>> ________________________________________
>> From: CCP4 bulletin board [[log in to unmask]] On Behalf Of Jacob Keller [[log in to unmask]]
>> Sent: Monday, December 06, 2010 12:15 PM
>> To: [log in to unmask]
>> Subject: Re: [ccp4bb] pH dependent conformational change
>>
>> Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive?
>>
>> Jacob
>>
>>
>> On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius <[log in to unmask]<mailto:[log in to unmask]>> wrote:
>> Daniel,
>>
>> You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes.
>>
>> You said "easy", so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too.
>>
>> And then there are the "difficult" ways...
>>
>> MM
>>
>>
>>
>>
>> On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote:
>>
>>
>> Dear CCP4 colleagues,
>>
>>
>>
>> We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks.
>>
>>
>>
>> As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks.
>>
>>
>>
>> Best,
>>
>> Daniel
>>
>>
>>
>> -----------------------------------------------------------------------
>> Mischa Machius, PhD
>> Director, Center for Structural Biology
>> Assoc. Professor, Dept. of Pharmacology
>> Member, Lineberger Comprehensive Cancer Center
>> University of North Carolina
>> 4079 Genetic Medicine
>> CB#7365
>> 120 Mason Farm Road
>> Chapel Hill, NC 27599-7365, U.S.A.
>> tel: +1-919-843-4485
>> fax: +1-919-966-5640
>> email: [log in to unmask]<mailto:[log in to unmask]>
>>
>>
>>
>>
>> --
>> *******************************************
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> cel: 773.608.9185
>> email: [log in to unmask]<mailto:[log in to unmask]>
>> *******************************************
>>
>
>
>
> --
> *******************************************
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: [log in to unmask]
> *******************************************
>
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: [log in to unmask]
*******************************************
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