Characterization of Protein Detergent Complexes

1
Characterization of Protein Detergent Complexes
Innokenty Maslennikov, Georgia Kefala, Casey
Johnson, Chris Dickson, Roia Katebian, Luis
Esquivies, Senyon Choe, Witek Kwiatkowski
2

• Topics
• Detergent analysis during protein production by
  NMR
• PDC analysis by analytical ultracentrifugation
  and light scattering

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Goals of Detergent Analysis by NMR
To determine

• optimal concentration of extraction detergent
• detergent exchange efficiency
• detergent amount during concentration by filters

FC14
9
22
3
2
I. Maslennikov, G. Kefala, C. Jonson, R. Riek, S.
Choe, W. Kwiatkowski BMC Structural Biology ,
774
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Determining optimal concentration of extraction
detergent

• Full extraction achieved
• with gt 90 detergent
• retained
• Depends on the protein
• yield

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Determining detergent exchange efficiency
FC14 --gt DDM
A FC14 is not exchanged
5x2cv Wash 0.5mM DDM
5x2cv Wash 5mM DDM 3x2cv Wash 0.5mM DDM
B FC14 is exchanged
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Determining detergent amount during concentration
by filters
Two types of filters Poliethersulfone
(Vivaspin)? Regenerated Cellulose (Amicon) Two
MW cutoffs 30KD 50KD Ten different
detergents FC14, DDM, ZW3-14, FC12, DM,
SDS, DPh, LDAO, NG, OG Vivaspin50 for lt 100kD PDC
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Goals of PDC analysis by analytical
ultracentrifugation and light scattering

• To check homogeneity of the sample
• To determine oligomeric state of the protein
• To determine protein-to-detergent ratio
• To investigate the effect of detergents used as
  additives for crystallization

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Analysis of homogeneity and protein aggregation
state by analytical ultracentrifugation
Mb Mp (1 ??p) Md (1 ??d)
Mpdc112.2 Md/Mp0.93
Mp83.3
Etk-M (FC12)?
EnvZ-M (DDM)?
DPh
FC14
Etk-M (DDM)?
Etk (FC12)?
EnvZ-M (FC14)?
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Analysis of homogeneity and protein aggregation
state by light scattering
RstB in FC12 (68 kDa)?
RstB in FC12 (68 kDa)?
PDC 137.3 kDa
RstB 70.1 kDa
FC12 67.2 kDa
A280 cPDC e l
e fp ep (1-fp) ed
dn/dc fp (dn/dc)p (1-fp) (dn/dc)d
RI cPDC (dn/dc)?
R? c M (dn/dc)2
M mass of the PDC
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Analysis of the effect the detergents used as
additives have on crystallization by light
scattering
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Characterization of Protein Detergent Complexes
Innokenty Maslennikov, Georgia Kefala, Casey
Johnson, Chris Dickson, Roia Katebian, Luis
Esquivies, Senyon Choe, Witek Kwiatkowski