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| Institute: |
Lab or Branch |
| NIAAA |
Laboratory of Membrane Biochemistry and
Biophysics |
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| Title: |
| Probing 3D structure of protein kinase
C by cross-linking and mass spectrometry |
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| Authors: |
| B. X. Huang, H-Y. Kim |
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| Abstract: |
| During activation, protein kinase C (PKC)
undergoes stepwise conformational changes. These 3D structural
changes ultimately relieve the pseudosubstrate domain
and expose catalytic domain for its activity. Therefore,
examining 3D structure of PKC is important to understand
the molecular mechanisms involved in PKC activation processes.
We demonstrate here an approach to probe the 3D structure
of PKCa, using chemical cross linking and mass spectrometric
analysis, with the assistance of O-18 labeling in identifying
cross-linked peptides. PKCa was cross-linked with BS3,
a lysine (K) specific crosslinker. The monomeric cross-linked
PKCa was isolated by SDS/PAGE and digested with trypsin
in pure water or 95% O-18 water. Alternatively the cross-linked
PKC was digested in solution after buffer exchange using
molecular centrifugal filtration or dialysis. Nano-electrospray
MS analysis was carried out on a high resolution PE-Sciex
Qq-TOF or an Agilent 1100 series LC/MSD ion trap mass
spectrometer equipped with a 2D-HPLC device. Newly emerging
cross-linked peptides were identified by comparing the
mass spectra of tryptic digest from the BS3 treated with
those from unmodified PKCa. The cross-linked peptides
were further identified with the assistance of O-18 labeling.
Cross-linking between two different peptide segments resulted
in an 8 amu shift in mass spectra, while unmodified tryptic
peptides or peptides with cross-linking within a peptide
segment showed a 4 amu shift, due to the incorporation
of two O-18 atoms in each C-terminus. This approach also
allowed the distinction of b ion series from y ions in
MS/MS spectra. More than 75% or 80% of the PKC sequence
was accounted by the MS/MS analysis of in-gel or in-solution
tryptic digests, respectively. The absence of the peptide
peak with mass 1993.880 indicated that the Arg-19 in the
pseudosubstrate was resistant to proteolysis when PKCa
was inactive. This implied that pseudosubstrate was "locked"
in the substrate-binding cavity. At least 10 cross-linked
sites were identified. K41, K62, and K76 in C1a domain,
K131 or K141, and K158 of C1b domain, K486 in the substrate-binding
domain, and K517 near the substrate-binding cavity participated
in cross-linking. Labeling with O-18 indicated that at
least one peptide was cross-linked between two different
segments. The mass spectrometric data from cross-linked
peptides will be presented along with the 3D-structural
information of PKC supporting the observed K cross-linking. |
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