Hi, we've had a similar situation: a protein-peptide complex with a Kd in the nM range crystallized in the same condition as the protein alone, and yielded a structure of a complex (voltage-gated calcium channel beta subunit). The exact crystal contacts turned out to be a bit different, as the peptide would clash with a neighbouring molecule in the lattice.
However, a mutant protein that increased the Kd to ca 160nM (as confirmed by ITC), using the same peptide crystallized in the same conditions, but this time not as a complex. This effect was reproducible: the WT consistently crystallized as complex, whereas relatively mild mutants (Kd in 100nM range and worse) only yielded crystals of the apo-protein. Conclusion would be that crystal contacts can break relatively tight protein-protein interactions in the ~100nM range, and that crystal contacts are not always that weak. However, the crystallization conditions themselves (PEGs, non-neutral pH) are likely to affect the binding as well. Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 10:42 AM, Philippe DUMAS <[EMAIL PROTECTED]> wrote: > Hello > > We have had an interesting example where the crystal packing seems to have > won against the "biological interaction". This is about a "sliding clamp" > made of a very symmetric homodimer having the shape of a ring (encircling > DNA during its replication). > This "beta-ring" had been crystallized alone by the Kuriyan group in P1 > (thus there was NCS). In our case, we crystallized it with an additional > peptide mimicking the C-term of a polymerase binding to the beta-ring > [Burnouf et al, JMB 335(2004) 1187]. We expected a symmetric binding of two > peptides/ring (one peptide for each protein in the dimer). However, we > observed only one peptide/ring. It turns out that we had obtained exactly > the same packing in P1 and that one of the two possible binding sites was > engaged in crystal contacts. We estimated the Kd of peptide-ring interaction > as being in the µmolar range and that there was only a few percent of > beta-rings in crystallization drops being singly occupied. Yet the > crystallization process selected this minor species to build crystals with > (supposedly) a good crystal packing, rather than "finding" another crystal > packing accomodating the doubly-occupied species present in large excess. > Our conclusion was that a very modest gain of ca. 2 kcal/mol in the free > energy of interaction of singly-occupied beta-rings was sufficient to > account for their selection to build crystals against a great majority of > doubly-occupied "contaminants". This is exactly the order of magnitude > mentioned by Ed Pozharski: a single additional H-bond is enough to account > for 2 kcal/mol ! And apparently this may be enough to win against > "biological interactions". Let us not forget that there are many processes > comparable to crystallization in living cell... > > I hope this story makes sense in the frame of this discussion. > > Philippe Dumas > IBMC-CNRS, UPR9002 > 15, rue René Descartes 67084 Strasbourg cedex > tel: +33 (0)3 88 41 70 02 > [EMAIL PROTECTED] > > -----Message d'origine----- > De : CCP4 bulletin board [mailto:[EMAIL PROTECTED] la part de Ed > Pozharski > Envoyé : Monday, June 30, 2008 4:50 PM > À : [email protected] > Objet : Re: [ccp4bb] Weakest protein-protein complex crystallised > > > The word "weak" is, of course, relative. Free energy of crystallization > is roughly 1-2 kcal/mole of crystal contacts (I think I carried this > number from Sir Blundell's book, but quick look at papers by Peter > Vekilov's group seems to confirm it - am I wrong on this?). I think > that crystal contacts are still much weaker than any interaction of > biological importance (perhaps I am wrong on this one too and there are > important biological protein-protein interaction with 10mM affinity, but > I doubt that they are many). > > On Mon, 2008-06-30 at 10:09 -0400, Patrick Loll wrote: > > I hope this isn't too much of a foray into philosophy and semantics, > > but can't you argue that the crystals themselves are weak complexes? > > And since the energies of crystal contacts are typically very weak, I > > would further argue that you should be able to crystallize ANY complex > > with an association constant corresponding to energies as low as those > > associated with crystal contacts. Of course, it's not guaranteed, any > > more than getting a crystal is guaranteed--you need some luck. > > > > > > Of course, it's Monday AM, and I haven't approached my asymptote for > > caffeination. Am I talking through my hat? > > > > > > Pat > > > > > > > > > > On 29 Jun 2008, at 3:36 PM, Derek Logan wrote: > > > Hi, > > > > > > > > > Can anyone advise me what is currently the weakest protein-protein > > > complex yet crystallised? Google searching turned up a paper from > > > the Tromsø crystallography group (Helland et al. 1999, JMB 287, 923– > > > 942) in which a complex between beta-trypsin and a P1 mutant of BPTI > > > with a Kd of 68 uM was described as belonging to the weakest > > > complexes solved to date, but this article was from 1999 and much > > > water has passed under the bridge since then. > > > > > > > > > Thanks > > > Derek > > > _________________________________________________________________ > > > Derek Logan tel: +46 46 222 1443 > > > Associate professor fax: +46 46 222 4692 > > > Molecular Biophysics mob: +46 76 8585 707 > > > > > > Centre for Molecular Protein Science > > > Lund University, Box 124, 221 00 Lund, Sweden > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > --------------------------------------------------------------------------------------- > > > > Patrick J. Loll, Ph. D. > > > > Professor of Biochemistry & Molecular Biology > > > > Director, Biochemistry Graduate Program > > > > Drexel University College of Medicine > > > > Room 10-102 New College Building > > > > 245 N. 15th St., Mailstop 497 > > > > Philadelphia, PA 19102-1192 USA > > > > > > (215) 762-7706 > > > > [EMAIL PROTECTED] > > > > > > > > > -- > Edwin Pozharski, PhD, Assistant Professor > University of Maryland, Baltimore > ---------------------------------------------- > When the Way is forgotten duty and justice appear; > Then knowledge and wisdom are born along with hypocrisy. > When harmonious relationships dissolve then respect and devotion arise; > When a nation falls to chaos then loyalty and patriotism are born. > ------------------------------ / Lao Tse / > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
