The impact of protein characterization in structural proteomics.
Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.
Geerlof
Arie
A
EMBL Hamburg Outstation, Notkestrasse 85, Hamburg, Germany. arie.geerlof@embl-hamburg.de
Brown
J
J
Coutard
B
B
Egloff
M P
MP
Enguita
F J
FJ
Fogg
M J
MJ
Gilbert
R J C
RJ
Groves
M R
MR
Haouz
A
A
Nettleship
J E
JE
Nordlund
P
P
Owens
R J
RJ
Ruff
M
M
Sainsbury
S
S
Svergun
D I
DI
Wilmanns
Matthias
M
D003460:Crystallization; D006868:Hydrolysis; D008027:Light; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012542:Scattering, Radiation; D014357:Trypsin; D014461:Ultracentrifugation; D014965:X-Rays
D011506:Proteins; D014357:Trypsin