[RASMB] AUC capabilities

Thu Jan 27 06:51:17 PST 2011

Hi, 

Isn’t there a correlation between, rotational correlation time and particle
shape and size?

R(t) = R0exp(-1/tr) where R(t) is the decay, R0 is the max initial
anisotropy and tr the rotational correlation time (motility)

And

Tr=nV/kT (Stokes-Einstein), where n is the viscosity, V is the hydrodynamic
volume for a spherical particle and kT is the Bolzmann constant and
Temperature.

In this case it would indeed not be radius of gyration but the hydrodynamic.

Anyway, I am certainly not certain about it and maybe this approach is too
complicated. 

Titus

From: Mattia Rocco [mailto:mattia.rocco at istge.it] 
Sent: Thursday, January 27, 2011 9:10 AM
To: Titus M. Franzmann; rasmb at server1.bbri.org
Subject: Re: [RASMB] AUC capabilities

Just a correction: from fluorescence anisotropy you don't get the radius of
gyration, but possibly the harmonic mean of the rotational correlation time.

As for using TEM with negative staining, one has to be absolutely sure that
adhesion on the grid doesn't influence the monomer-dimer equilibrium...

Mattia

At 08:52 AM 1/27/2011 -0500, Titus M. Franzmann wrote:

Ewa,
Typically one tries to span a protein concentration range of at least 5times
lower and 5times greater the kDa. Optimally 10times.
So you would go at least from 2.5 to about 125 nM. Given the already low
signal intensity of 0.1 at 220 at 25 nM you may not be able to observe the
fully dissociated particle (monomers) and thus may not be able to acquired a
dataset with a fully established monomer baseline. That would give an
uncertainty in the Kd when fitting it to a mono-dimer association model
(assuming no equilibrium intermediate populates).
Anyway, I have carried out a couple of experiments at low signals, e.g. 0.05
Abs. and determining the S-value is fairly robust, but fitting these low
signal datasets to find proper Mw values is fairly difficult. Of course one
can gain confidence by running triplicates and consolidate the datasets in a
global fit. From this you can at least estimate what kind of particles you
have. If the system is a fast interacting system, you should be able to use
the Sw20avg values and plot them as the function of the protein
concentration. It will be difficult to do this, when the system is slow
interacting and the signal gets separated and distributed among the monomer
and dimer fractions.
Anyway, have you considered fluorescence anisotropy? According to your
information, the protein contains several trp residues that you can use to
monitor the assembly process. From the fluorescence anisotropy you can also
gain information about the radius of gyration and make some estimate about
the shape and size. Alternatively, we have recently used TEM to visualize
monomers and dimers of a 39 kDa monomer. Surprisingly, we got some
information using negative staining techniques and particle population
analysis. You would at least get some idea about what you get.
Titus

Dr. Titus M. Franzmann
S. G. Walter Lab
Mol., Cell. and Develop. Biol. Dept. 
University of Michigan
4140C Nat. Sci. Bldg
830 N. University Ave.
Ann Arbor, MI 48109-1048

-----Original Message-----
From: rasmb-bounces at server1.bbri.org [mailto:rasmb-bounces at server1.bbri.org]
On Behalf Of Leech, AP
Sent: Thursday, January 27, 2011 8:17 AM
To: rasmb at server1.bbri.org
Subject: Re: [RASMB] AUC capabilities

Hi All,

Eva, if you put all 50 ug into 120 uL for an equilibrium experiment,
this would be ~0.4 mg/ml, or 7 uM, giving an approx absorbance of
0.4 at 280 nm. If your Kd is right, this would practically all be
dimer. To get down to sub-Kd concentrations, as Borries says, the
absorbances would be impractically low.

If you put it all into 400 uL for a velocity experiment, the initial
concentration would be 0.125 mg/ml, 2.1 uM, absorbance ~0.13 at 280nm.
This is well above the Kd also, I will leave someone with more
experience to say if dissociation would affect the boundary detectably,
I suspect not.

It might be worth considering SPR. If you can attach one monomer to
the chip surface without compromising dimerisation (practically I think
you would attach the dimer and look for dissociation; there might be
a problem with both halves of the dimer attaching), then wait for
dissociation and flow various concentrations over the surface. The
amounts are more in the ball park for what you have (say 10 ug for
immobilisation and a few ug for each trial of free protein) though
it's still not a lot! and you need to allow for control experiments
for non-specific binding etc.

Best regards,

Andrew

On 27/01/2011 04:29, Ewa Folta-Stogniew wrote:
> At 10:39 PM 1/26/2011, you wrote:
>> >
>> > Hello,
>> >
>> > is it possible to determine dimerization constant in the order of 25
>> > nM using AUC? What will be the minimal amount of protein needed?
>> >
>> > Ewa
>> >
>>
>> Hi Ewa,
>>
>> the answer to your question is: it depends on several factors...
>>
>> 1. what optical system do you plan to use - there are choices:
>> absorbance, Rayleigh interference, fluorescence emission
>
> absorbance or interference
>
>
>> 2. If you use absorbance, there is a question about the extinction
>> coefficient and the wavelength. Depending on extinction, 25 nM may
>> be well within reach at several detection wavelengths.
>
> A280(0.1%)=0.85 (mg-1mL cm-1)
>
>> 3. Related to (2) is the question of size - the larger the protein,
>> presumably the larger the extinction coefficient (more trp, tyr residues
>> at 280 nm, more peptide backbone absorbance at lambda <= 230 nm). Ditto
>> for Rayleigh interference - the larger the protein, the more refraction
>> you will see and 25 nM could be well within range.
>
>
> 59 kDa monomer
>
>
>> 4. If you consider using the fluorescence detector, then 25 nM label
>> of the right excitation/emission should be well within the range of
>> detection.
>
> no fluorescence detection available
>
>
>> 5. in order to get accurate Kds you would want to measure ideally
>> at concentrations above and below the Kd to get good signal
>> of the monomer and the dimer, so multiple concentrations measured
>> either in velocity or equilibrium mode and globally fitted should
>> give you the desired information. Related to that is that the
>> Kd needs to be in the range where you are measuring. If your Kd is
>> 100 micromolar and you are measuring at 25 nM and below, your answer
>> is going to be quite unreliable. At the same token, measuring at
>> 100 uM when your Kd is at 20 nM will not give you the desired result.
>
> Assuming Kd of 25 nM and the extinction coefficient as above- can one
> determine Kd for dimerization from 50 micrograms of protein total (25
> micrograms preferred)?
>
> If this is theoretically feasible, does anybody know of any published
> AUC results for similarly low Kd?
>
> thanks,
>
> Ewa
>
>
>> Your question about how much protein is needed at a minimum depends
>> also on the questions I posed above. If you provide additional
>> information we may be able to tell you more specifically for your sample.
>>
>> Regards, -borries
>
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-- 
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Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
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