Hi Sally,

Crystallization of proteins with multiple phosphorylation sites is often a difficult problem. Like you said it is often an issue of inhomogeneity. Several suggestions that may be helpful:

First, I would use a bit of your protein sample for Mass Spec analysis. It is possible (though maybe not likely if your protein has multiple sites) that your sample is homogenous and you don't even have to worry. 

Next, I would think about what you are trying to learn about this protein from the crystal structure. If you are interested in how the structure is effected by phosphorylation, there are several things you can do. To obtain a structure of the fully dephosphorylated protein you can use a nonspecific phosphatase before crystallization to remove all the phosphates. Alternatively you can mutate the phosphorylation sites. Mutating Ser to Ala, Thr to Val, and Tyr to Phe will probably not effect your protein's native structure very much (if at all), no matter how many substitutions you have to make. You should, however, determine the phosphorylation sites beforehand so you are not unnecessarily mutating S,T, and Y residues that aren't ever going to be phosphorylated. To learn how phosphorylation of Ser/Thr residues influences the structure, it is often useful to mutate the phosphorylation site to Glu, because it's size, shape, and charge properties are not very different from phosphoserine or phosphothreonine. This strategy is not perfect, but it can work well, especially since it directly addresses the problem of inhomogeneity.

A final suggestion is to just try and crystallize your protein with inhomogeneous phosphorylation. Depending on how inhomogeneous your sample is at each site, you will likely see phosphates that have only partial occupancies (and you might not ever see phosphates at sites that are weakly phosphorylated), but it still might be sufficient to gain the information you are interested in. Again, if you plan to do this, mass spec may help you understand your data. If you can mass spec digested protein you may be able to figure out which phosphorylation sites have high and low occupancy (I'm not a mass spec expert though), and you could compare this data with what you observe in your crystal structure.

Another quick thing to mention is to be weary of your expression system. If you express your protein in an exogenous expression system, the phosphorylation state you observe may be different (dramatically different in some cases) from what occurs in the native context, which is what you're interested in. Again, mass spec could help you here.

Hope this helps,

Mike Thompson





----- Original Message -----
From: "Sally Pham Thanh Van" <[log in to unmask]>
To: [log in to unmask]
Sent: Wednesday, November 10, 2010 11:03:49 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Crystal structures of phosphorylated proteins

To make it clearer, I'm looking for successful examples of
crystallization of phosphorylated proteins, especially ones containing
multiple phosphorylation sites, and would like to access
crystallizability of these protein types. I want to produce and
crystallize protein at its phosphorylation state but I learned
phosphorylation may cause inhomogeneity problem and interfere with
crystallization. It's not a good option to replace these sites anyway
because there are many and may affect native structure of the protein.

Any input would be appreciated,
Sally.







On Wed, Nov 10, 2010 at 1:26 PM, Atlanta Cook <[log in to unmask]> wrote:
> Perhaps you might pose a more directed question... Do you want to produce
> phosphorylated protein? Are you having trouble crystallising a protein that
> is phosphorylated?
>
>
> Atlanta
>
>
>
>
> On 10 Nov 2010, at 11:09, Sally Pham Thanh Van wrote:
>
>> Dear all,
>>
>> Could you please tell me any information regarding to crystal
>> structures of phosphorylated proteins? Your input would be
>> appreciated.
>>
>> Best regards,
>> Sally.
>
>

-- 
Michael C. Thompson

Graduate Student

Biochemistry & Molecular Biology Division

Department of Chemistry & Biochemistry

University of California, Los Angeles

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