EMBO Global Exchange Lecture Course 3 May 2011 Beijing China Quantitative characterization Quantitative characterization of mixtures and complex fo mation formation Peter Konarev European Molecular Biology Laboratory, Hamburg Outstation BioSAXS group
Scattering from mixtures Scattering from monodisperse g p ( h (shape polydispersity) l di it ) systems π ∑ ∑ = D I ( s ) v I ( s ) sin sr ∫ ∫ = I ( s ) 4 p ( r ) dr k k sr 0 k The scattering is proportional to that The scattering is proportional to that of a single particle averaged over all For equilibrium and non-equilibrium orientations, which allows one to mixtures solution scattering permits to mixtures, solution scattering permits to determine size, shape and internal determine the number of components structure of the particle at low ( 1-10 and, given their scattering intensities and, given their scattering intensities nm ) resolution. ) l ti I k (s), also the volume fractions
Outlines Polydisperse & interactive systems in ATSAS Equilibrium oligomeric mixtures (OLIGOMER) Assembly/disassembly processes (SVDPLOT, MIXTURE) Natively unfolded proteins and Natively unfolded proteins and multidomains proteins with flexible linkers (EOM, Weifeng Shang lecture) Applications of ATSAS for biological studies Oligomerization tuned by protein/salt concentration Oligomerization tuned by protein/salt concentration Multiple assembly forms Temperature dependent transitions
Program OLIGOMER for SAXS analysis Program OLIGOMER for SAXS analysis Input parameters: 1) experimental data file (ASCII file *.dat) 2) form-factor file with the scattering from the components (can be easily prepared by FFMAKER) (can be easily prepared by FFMAKER) ∑ ∑ = I ( ( s ) ) v I ( ( s ) ) k k k k k Output parameters: 1) the fit to experimental data (* fit file) Output parameters: 1) the fit to experimental data (*.fit file) 2) the volume fractions of the components (in oligomer.log) OLIGOMER can be launched in batch mode for multiple data sets: oligomer.exe /ff formfactor.dat /dat hp*.dat /un 2 /smax 0.25 Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. (2003) J. Appl. Cryst. 36 , 1277
FFMAKER as pre-tool for OLIGOMER To quickly create form-factor file from pdb files and/or from scattering data files (either from ASCII *.dat files or g ( from GNOM output files where desmeared curve will be taken for intensity) Batch mode: ffmaker 1.dat 2.dat /undat 2 3.out /unout 2 ffmaker *.pdb m1.dat /smax 0.3 /ns 201 /lmmax 20 ffmaker 6lyz.pdb *.dat /sgrid m2.dat ffmaker ALL ffmaker ALL all data files with "pdb", "ent", "out" or "dat" extension will be taken,
Oligomer content in mixtures lg I, relative Monomer/dimer equilibrium of Drosophila kinesin (1) -1 (2) (2) -2 -3 (3) Volume fraction -4 -4 (4) (4) 1.0 1 0 Monomer -5 (5) Dimer -6 (6) ( ) 0.5 -7 (7) -8 (8) (8) -9 0.0 0 2 4 6 8 10 12 0 1 2 s, nm-1 c, mg/ml Kozielski, F ., Svergun, D.I., Zaccai, J. Wade, R.H. & Koch, M.H.J. (2001) J. Biol. Chem. 276 , 1267
Momomer/dimer equiilbrium in tetanus toxin Electrophoresis, size exclusion Monomeric fraction fraction chromatography chromatography and mass Dimeric spectrometry fraction reveal concentration- t ti dependent oligomerization Mixtures of the receptor of the receptor binding H(C) domain of tetanus toxin Ab initio and rigid body analysis of the dimeric H(C) domain using the structure of the monomer in the crystal (1FV2) and accounting that the mutant Cys869Ala remains always monomeric yield a unique model of the dimer Qazi, O., Bolgiano, B., Crane, D., Svergun, D.I., Konarev, P .V ., Yao, Z.P ., Robinson, ., Brown, K.A. & Fairweather N. (2007) J Mol Biol. 365 , 123–134. C.V
Oligomeric state of Tricorn protein in solution Tricorn protease is a major component in the cleavage of oligopeptides produced by the proteasome. Tricorn appeared to be a multifaceted system in solution. The estimated molecular mass of the particles (380 kDa) was significantly lower than the theoretical value of 720 kDa tricorn hexamer, suggesting partial tricorn hexamer suggesting partial dissociation of the tricorn hexamers in solution. SAXS data were fitted by a linear combination of the scattering from tricorn monomers (53%), dimers (14%) and hexamers (33%) using OLIGOMER hexamers (33%) using OLIGOMER. Goettig, P., Brandstetter, H., Croll, M., Gohring, W., Konarev, P.V., Svergun, D.I, Huber, R., and Kim, J.S. (2005) J Biol Chem. 280 , 33387-33396
Studies of adrenodoxin (Adx) : cytochrome c (C c ) complex by SAXS and NMR Adx is involved in steroid hormone biosynthesis by acting as an electron shuttle between adrenodoxin reductase and cytochromes. Solutions of native (WT) and cross-linked (CL) complex of C c and Adx were measured by SAXS at different conditions: a) solute concentration range from 2.4 to 24.0 mg/ml; b) 10 mM Hepes / 20mM potassium phosphate (pH 7 4) buffer; b) 10 mM Hepes / 20mM potassium phosphate (pH 7.4) buffer; c) with addition of NaCl (from 0 up to 300 mM). Adx Each protein has Molecular Mass (MM) of about 12.5 kDa. Each protein has Molecular Mass (MM) of about 12.5 kDa. For CL complex C c V28C and AdxL80C mutants were linked by a disulfide bond. X. Xu, W. Reinle, F. Hannemann, P. V. Konarev, D. I. Svergun, R. Bernhardt & M. Ubbink JACS (2008) 130 , 6395-6403 ¶ Cc
Studies of (Adx) : (C c ) complex formation CL Complex CL Complex DAMMIN and SASREF models The experimental scattering from the CL complex does not depend on the solute concentration and addition of NaCl. It is compatible with 1:1 complex. X. Xu, W. Reinle, F. Hannemann, P. V. Konarev, D. I. Svergun, R. Bernhardt & M. Ubbink JACS (2008) 130 , 6395-6403 ¶
Studies of (Adx) : (C c ) complex formation Native Complex Native Complex Conc=4.8 mg/ml, 200 mM NaCl DAMMIN and SASREF models Conc=24 mg/ml No salt The native complex strongly depends on the sample concentration and on the amount of NaCl in the buffer. At high protein concentration it forms heterotetramer with 2:2 stoichiometry, whereas at high salt concentration it dissociates into two individual proteins. proteins. X. Xu, W. Reinle, F. Hannemann, P. V. Konarev, D. I. Svergun, R. Bernhardt & M. Ubbink JACS (2008) 130 , 6395-6403 ¶
Studies of (Adx) : (C c ) complex formation Native Complex Native Complex lgI, relative Native complex, no salt CL 4 complex (1) (1) c,mg/ml 24 12 6 2.4 3-12 3 (2) 2 28.3 ± 0.7 28.3 ± 0.7 26.5 ± 0.5 24.4 ± 0.7 21.4 ± 0.5 R g , Å (3) 1 90 ± 5 90 ± 5 90 ± 5 80 ± 5 80 ± 5 D max , Å 0 (4) V p , 10 3 Å 3 63 ± 6 52 ± 5 43 ± 5 35 ± 4 42 ± 5 -1 (5) 44 ± 5 42 ± 5 35 ± 4 25 ± 4 22 ± 3 -2 2 MM kD MM, kDa 44 5 42 5 35 4 25 4 22 3 -3 6 ± 5 24 ± 5 V mon,% 0 0 0 -4 8 ± 5 8 ± 5 25 ± 5 25 ± 5 24 ± 5 24 ± 5 0.1 0.2 0.3 0.4 V dim,% V 0 0 100 100 s, A-1 o 48 ± 5 47 ± 5 54 ± 5 52 ± 5 V tri,% 0 OLIGOMER fits 52 ± 5 45 ± 5 15 ± 5 V tet,% 0 0 tet,% X. Xu, W. Reinle, F. Hannemann, P. V. Konarev, D. I. Svergun, R. Bernhardt & M. Ubbink JACS (2008) 130 , 6395-6403 ¶
Studies of adrenodoxin (Adx) : cytochrome c (C c ) complex by SAXS and NMR NMR structure of CL complex NMR structure of CL complex overlaps well with SAXS model. The ensemble of native Adx:Cc complex structures from the PCS simulation. Oligomerization behavior of the native complex in solution indicates a stochastic nature of complex formation. The native Adx/C c is entirely dynamic and can be considered as a pure encounter complex. X. Xu, W. Reinle, F. Hannemann, P. V. Konarev, D. I. Svergun, R. Bernhardt & M. Ubbink JACS (2008) 130 , 6395-6403 ¶
Solution structure of human Pex5/Pex14/PTS1 protein complexes obtained by SAXS protein complexes obtained by SAXS The Pex5p import receptor DAMMIN and BUNCH models of Pex5p recognizes peroxisomal matrix g p proteins with C-terminal peroxisomal targeting signal (PTS). After docking to protein complexes on the membrane these proteins are translocated across the membrane. The interaction of the cargo- loaded Pex5p receptor and the loaded Pex5p receptor and the peroxisomal membrane protein The free full length human Pex5p is Pex14p is the essential primary monomeric in solution, with an elongated, docking step . partially unfolded N-terminal domain. p y Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284 , 25334-25342
Solution structure of human Pex5/Pex14/PTS1 protein complexes obtained by SAXS protein complexes obtained by SAXS Titration studies yielded Titration studies yielded a 1:6 stoichiometry for the Pex5p/Pex14p complex Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284 , 25334-25342
Solution structure of human Pex5/Pex14/PTS1 protein complexes obtained by SAXS protein complexes obtained by SAXS DAMMIF and SASREF models of ternary complex Inter subunit contacts were imposed for Pex14p(N) interactions with the WxxxY/F motifs of Pex5p(F) based on NMR data based on NMR data Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284 , 25334-25342
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