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

2. Topics: Detergent analysis during protein production by NMR
PDC analysis by analytical ultracentrifugation and light scattering 2

3. 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 , 7:74 3

4. Determining optimal concentration of extraction detergent
Full extraction achieved
with > 90% detergent
retained
Depends on the protein
yield 4

5. Determining detergent exchange efficiency
FC14 --> DDM
A: FC14 is not
exchanged
5x2cv Wash
0.5mM DDM
B: FC14 is
exchanged
5x2cv Wash
5mM DDM
3x2cv Wash
0.5mM DDM 5

6. 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 < 100kD PDC

7. 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

8. Mb = Mp (1 – ρῡp) + Md (1 – ρῡd)
Analysis of homogeneity and protein aggregation state by analytical ultracentrifugation
Mb = Mp (1 – ρῡp) + Md (1 – ρῡd)
Mpdc=112.2
Md/Mp=0.93
Mp=83.3
Etk-M
(FC12)‏
EnvZ-M
(DDM)‏
DPh
FC14
Etk
(FC12)‏
Etk-M
(DDM)‏
EnvZ-M
(FC14)‏ 8

9. dn/dc = fp (dn/dc)p + (1-fp) (dn/dc)d
Analysis of homogeneity and protein aggregation state by light scattering
RstB in FC12 (68 kDa)‏
RstB in FC12 (68 kDa)‏
PDC kDa
RstB
70.1 kDa
FC12
67.2 kDa
A280 = cPDC ε l
ε = fp εp + (1-fp) εd
dn/dc = fp (dn/dc)p + (1-fp) (dn/dc)d
RI = cPDC (dn/dc)‏
Rθ ≈ c M (dn/dc)2
M – mass of the PDC 9

10. Analysis of the effect the detergents used as additives have on crystallization by light scattering

11. Characterization of Protein Detergent Complexes
Innokenty Maslennikov, Georgia Kefala, Casey Johnson, Chris Dickson, Roia Katebian, Luis Esquivies, Senyon Choe, Witek Kwiatkowski 11