Re: [ccp4bb] relationship between B factors and Koff

2010-11-19 Thread Narayanasami Sukumar 
Sebastiano,
We observed similar phenomenon in our protein-protein complex.  This
reference will give you details (Crystal Structure of an Electron Transfer
Complex between Aromatic Amine Dehydrogenase- Azurin from Alcaligenes
faecalis, Biochemistry, 45, 13500-13510, 2006).

Sukumar

Narayanasami Sukumar
NE-CAT, Building 436E
Advanced Photon Source
Argonne National Laboratory
9700 S.Cass Ave
Argonne, IL 60439-4800
USA

Tel: 630-252-0681
Fax: 630-252-0687
e-mail: suku...@aps.anl.gov






On Fri, Nov 19, 2010 at 6:58 AM, Sebastiano Pasqualato 
sebastiano.pasqual...@ifom-ieo-campus.it wrote:

 Hi all,
 I have a crystallographical/biochemical problem, and maybe some of you guys
 can help me out.

 We have recently crystallized a protein:protein complex, whose Kd has been
 measured being ca. 10 uM (both by fluorescence polarization and surface
 plasmon resonance).
 Despite the 'decent' affinity, we couldn't purify an homogeneous complex in
 size exclusion chromatography, even mixing the protein at concentrations up
 to 80-100 uM each.
 We explained this behavior by assuming that extremely high Kon/Koff values
 combine to give this 10 uM affinity, and the high Koff value would account
 for the dissociation going on during size exclusion chromatography. We have
 partial evidence for this from the SPR curves, although we haven't actually
 measured the Kon/Koff values.

 We eventually managed to solve the crystal structure of the complex by
 mixing the two proteins (we had to add an excess of one of them to get good
 diffraction data).
 Once solved the structure (which makes perfect biological sense and has
 been validated), we get mean B factors for one of the component (the larger)
 much lower than those of the other component (the smaller one, which we had
 in excess). We're talking about 48 Å^2 vs. 75 Å^2.

 I was wondering if anybody has had some similar cases, or has any hint on
 the possible relationship it might (or might not) exist between high a Koff
 value and high B factors (a relationship we are tempted to draw).

 Thanks in advance,
 best regards,
 ciao
 s


 --
 Sebastiano Pasqualato, PhD
 IFOM-IEO Campus
 Dipartimento di Oncologia Sperimentale
 Istituto Europeo di Oncologia
 via Adamello, 16
 20139 - Milano
 Italy

 tel +39 02 9437 5094
 fax +39 02 9437 5990

Re: [ccp4bb] how to change a membrane protein into a water soluble protein?

2007-12-04 Thread Narayanasami Sukumar 
Another reference:

N. Sukumar, Y.Xu, D.L. Gatti, B.Mitra and F.S. Mathews
Structure of an Active Soluble Mutant of the Membrane-Associated
(S)-Mandelate Dehydrogenase
Biochemistry 40,9870-9878 (2001).

In this paper, the membrane protein is converted into the soluble
protein by replacing an intra-chain integral membrane-binding portion
(39 residues) by a segment from its closely related soluble homologue
(20 residues) and crystallized without any detergents.

Sukumar
-- 
N. Sukumar
NE-CAT, Building 436E
Argonne National Laboratory
9700 South Cass Avenue
Argonne, IL 60439
e-mail:  [EMAIL PROTECTED]

Tel: 630-252-0681
Fax: 630-252-0687




On Tue, 2007-12-04 at 08:06, Scott Pegan wrote:
 Don't know if anyone has mentioned this paper but its an exact example how
 to make a K channel soluble.
 
 Roosild TP, Choe S.
 
 Redesigning an integral membrane K+ channel into a soluble protein.
 Protein Eng Des Sel. 2005 Feb;18(2):79-84. Epub 2005 Mar 23.
 PMID: 15788421 [PubMed - indexed for MEDLINE]
 
 Scott
 
 
 On Tue, December 4, 2007 4:04 am, Brenda Patterson wrote:
  Another option is refolding which can increase soluble protein content and
  is
  used routinely to achieve soluble protein such as the TIMPs
 
  http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035
 
  http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck
 
 
  that said, this is not true of all membrane proteins.
 
  Addition of a fusion partner, MBP, to the normally membrane associated
  FMO3 has
  been shown to generate stable, soluble protein and the addition of a
  fusion
  protein allows purification downstream more easily.
 
  Here is a paper where they did as the original poster suggested and tried
  mutagenesis of hydrophobic regions, including a truncation of a membrane
  anchor.  They achieved increased solubility with this in combination with
  use
  of detergents.
 
  Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB,
  Williams DE.
  Abstract
  C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2
  enhances solubility.
  Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29.
 
 
  cheers
 
 
 
 
 
 
 
 
 
 
  Quoting Bil Clemons [EMAIL PROTECTED]:
 
  There is also the soluble KcsA.
 
  Computational design of water-soluble analogues of the potassium channel
  KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado
  (2004) PNAS 101, 1828-1833
 
 
  Bil
 
  
  Bil Clemons, PhD
  Assistant Professor of Biochemistry
  Caltech
  157 Broad Center
  MC 114-96
  Pasadena, CA 91125
  (626) 395-1796
  [EMAIL PROTECTED] mailto:[EMAIL PROTECTED]
  
 
 
 
 
  From: Thomas J Magliery PhD [EMAIL PROTECTED]
  Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED]
  Date: Mon, 3 Dec 2007 16:50:03 -0500
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: Re: [ccp4bb] how to change a membrane protein into a water
  solub=
  le
  protein?
  =20
  It's hard. See:
  =20
  J Mol Biol. 2005 May 6;348(3):777-87.
  X-ray structure of a water-soluble analog of the membrane protein
  phospholamban:=20
  sequence determinants defining the topology of tetrameric and
  pentameric
  coiled
  coils.
  Slovic AM, Stayrook SE, North B, Degrado WF.
  =20
  Slovic, A. M., Summa, C. M., Lear, J. D.  DeGrado,
  W. F. (2002). Computational design of a water-soluble
  analog of phospholamban. Protein Sci. 12, 337=AD348.
  =20
  Li, H., Cocco, M. J., Steitz, T. A.  Engelman, D. E.
  (2001). Conversion of phospholamban into a soluble
  pentameric helical bundle. Biochemistry, 40,
  6636=AD6645.
  =20
  Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S.,
  Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution
  structure of phospholamban: design of
  soluble transmembrane domain mutants.
  Biochemistry, 39, 6825=AD6831.
  =20
  Tom
  =20
  =20
  Daniel Jin wrote:
  Hi,
  I am wondering whether there is a way to turn a membrane protein with
  known crystal structure into a water soluble protein by systematic
  mutagenesis. I guess it should be doable if we introduce enough
  hydrophilic residues on the surface. Has anyone tested this crazy idea
  before? Thank you for your help.
  Best,
  Chen
  =20
  
  Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try
  it now.=20
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  2sR8H
  DtDypao8Wcj9tAcJ%20
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  =20
  --=20
  Thomas J. Magliery, Ph.D.
  Assistant Professor
  Department of Chemistry
   Department of Biochemistry
  The Ohio State University
  1043 Evans Laboratory
  100 West 18th Ave.
  Columbus, OH 43210-1185
  =20
  (614) 247-8425 office
  (614) 292-1685 fax
  [EMAIL PROTECTED]
  http://www.chemistry.ohio-state.edu/~magliery
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