[RASMB] Help with Small Self-Associating Peptide

[Tom Laue]

Hi Peter-
I would echo Holger's comment on vbar. There are too few amino acids to 
permit the 'averaging' that a calculated vbar relies on for accuracy. If 
there are many charged residues, the actual vbar will be lower than the 
calculated value, regardless of what the net charge is. A 1% variation 
in vbar will show up as a 3% variation in the molar mass, so be prepared 
to let the monomer mass float, and don't be surprised to find that it 
differs (typically is higher) than what you expect.
Best wishes,
Tom

On 12/12/2011 4:10 AM, HGSR (Holger Martin Strauss) wrote:
> Hi Peter,
> in addition to the comments by John, some more from my experience, 
> mainly from SE:
> - purity is absolutely essential, esp. when working with synthetic 
> peptides. Ideally, measure the integrity of the peptide with MALDI-TOF 
> before and after the experiment.
> - if at all possible, measure the vbar, the true value of your peptide 
> will likely deviate from the one Sednterp gives you. If you're only 
> after the stoichiometry of the complex, it might be sufficient to 
> stick with sigma (the lower limit of which you can define from the 
> data) and ratios thereof.
> - you can check the reversibility of your reaction from plots of 
> lnC/r2 at different starting concentrations.
> - the stoichiometry of the complex is more a problem of accuracy, so 
> you need the best data you can get; scan many replicates, use long 
> solution columns (200 microL and more), clean your lamp before the 
> experiment, use thoroughly dialysed buffer (2800 g/mole can be tough, 
> though). The decision wether your smallest sigma is really a monomer 
> or a dimer is easier to answer than wether you have a hexa- or a heptamer.
> - try to measure the baseline absorbance. If you do have a hexamer, 
> you should be able to clear the meniscus with long solution columns at 
> the highest speeds. You can get the baseline for all wavelengths used 
> from this condition.
> - if you can clear the meniscus, interference has clear advantages 
> over absorbance (better S/N, mostly) and will help you to define the 
> upper limit of the association. Be careful to measure a good water blank.
> - use a buffer of sufficient ionic strength. Some peptides carry 
> surprisingly high charge densities.
> - if the peptide unfolds at low concentrations, degradation can be a 
> real problem. If so, excluding as far as possible all sources of 
> microbial contamination can help, including rinsing your cells with 
> 70% ethanol.
> Best, Holger
>
> ------------------------------------------------------------------------
> *From:* rasmb-bounces at rasmb.bbri.org 
> [mailto:rasmb-bounces at rasmb.bbri.org] *On Behalf Of *Peter Edward 
> Prevelige Jr
> *Sent:* Friday, December 09, 2011 11:57 PM
> *To:* rasmb at rasmb.bbri.org
> *Subject:* [RASMB] Help with Small Self-Associating Peptide
>
> Hi All –
>
> Can you give me some advice on the best way to analyze a small (~2800 
> Da) peptide that we think is self associating. The CD shows increasing 
> helicity with concentration which continues to increase even at 10 
> mg/ml meaning we will likely have to record data at a less than ideal 
> wavelength (240 nm) Our interest is really in determining the 
> stoichiometry of the species formed at high concentration (we suspect 
> a hexamer), we care less about Kd. Looking through the archives I’ve 
> seen conflicting suggestions, (ie. sed velocity vs sed equilibrium). I 
> was thinking about mimicking the approach in the Braswell/Holtzer 
> paper but the peptide is somewhat valuable and limited so I wanted to 
> ascertain whether or not this was the optimal way to proceed.
>
> Thanks!
>
> Peter
>
> Peter E. Prevelige Jr.
>
> Professor
>
> Dept. of Microbiology, BBRB 416/6
>
> Univ. of Alabama @ Birmingham
>
> 845 19th St. South
>
> Birmingham AL. 35294-2170
>
> Phone 205 975-5327
>
> FAX 205 975-5479
>
> prevelig at uab.edu <mailto:prevelig at uab.edu>
>
> http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm
>
>
>
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
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E-mail: Tom.Laue at unh.edu
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