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proteins in membranes is actively driven by the lipid anchors of Ministerio de Economia and Competitividad (grant CTQ2013-
the proteins either through electrostatic interactions22,23 or 44334-P). We acknowledge support of the publication fee by the
through the hydrophobic effect (hydrophobic mismatch).17 CSIC Open Access Publication Support Initiative through its
Our results reveal that unsaturation in the acyl chain can also Unit of Information Resources for Research (URICI).
influence the clustering behavior of lipidated Ras proteins
owing to the decreased packing efficiency of the unsaturated
acyl chain in the lipid matrix. As a consequence of the perturbation
Conflicts of interest
of lipid packing, the tendency toward clustering increases, which
reduces the overall free energy of the proteolipid system.
There are no conflicts to declare.
To reveal also the effect of saturation of the acyl chain on the
insertion kinetics of the lipidated proteins, we performed a
fluorescence resonance energy transfer (FRET) based kinetics
assay. Large unilamellar vesicles (LUVs) composed of DOPC/
Notes and references
1 L. H. Chamberlain and M. J. Shipston, Physiol. Rev., 2015, 95, 341.
2 O. Quehenberger, A. M. Armando, A. H. Brown, S. B. Milne, D. S.
Myers, A. H. Merrill, S. Bandyopadhyay, K. N. Jones, S. Kelly, R. L. Shaner,
DPPC/chol (1 : 2 : 1) were labelled with 0.1 mol% rhodamine
DHPE. Insertion and binding of Bodipy labelled N-Ras protein
led to an increase in the FRET intensity as a function of time. The
kinetic data were found to be best fitted using a biexponential
association kinetics, i.e. insertion of the N-Ras protein proceeds via a
two-step process (the corresponding rate constants are summarized
in Fig. 3C). This is in agreement with previous studies which
proposed the two steps to be the insertion of the acyl chain into
the membrane which is followed by lateral rearrangement of the
protein in the membrane plane to attain the most stable equili-
brium state.24 Interestingly, the kinetic rate constants of both the
steps were found to be significantly higher for the unsaturated
N-Ras HDD/far than for the saturated N-Ras HD/far protein,
indicating a decreased activation energy for the insertion and
the lateral rearrangement process. This may be attributed to the
C. M. Sullards, E. Wang, R. C. Murphy, R. M. Barkley, T. J. Leiker,
C. R. Raetz, Z. Guan, G. M. Laird, D. A. Six, D. W. Russell, J. G. McDonald,
S. Subramaniam, E. Fahy and E. A. Dennis, J. Lipid Res., 2010, 51, 3299.
3 R. Takada, Y. Satomi, T. Kurata, N. Ueno, S. Norioka, H. Kondoh,
T. Takao and S. Takada, Dev. Cell, 2006, 11, 791.
4 X. Q. Liang, Y. Lu, T. A. Neubert and M. D. Resh, J. Biol. Chem., 2002,
277, 33032.
5 L. Muszbek, G. Haramura, J. E. Cluette-Brown, E. M. Van Cott and
M. Laposata, Lipids, 1999, 34, S331.
6 K. Brett, L. V. Kordyukova, M. V. Serebryakova, R. R. Mintaev,
A. V. Alexeevski and M. Veit, J. Biol. Chem., 2014, 289, 34978.
7 B. Zheng, G. K. Jarugumilli, B. Chen and X. Wu, ChemBioChem,
2016, 17, 2022.
8 F. Mohammadzadeh, V. Hosseini, A. Mehdizadeh, C. Dani and
M. Darabi, IUBMB Life, 2018, 71, 340.
9 N. Sorek, A. Akerman and S. Yalovsky, Methods Mol. Biol., 2013,
1043, 121.
10 I. S. Rosenfeld, G. D’Agnolo and P. R. Vagelos, Anal. Biochem., 1975,
64, 221.
11 F. Vernon and J. H. Khorassani, Talanta, 1978, 25, 410.
different oligomerization states of the N-Ras isoforms in the 12 C. Sarkar, G. Chandra, S. Peng, Z. Zhang, A. Liu and A. B. Mukherjee,
Nat. Neurosci., 2013, 16, 1608.
13 G. Liu, J. K. Lynch, J. Freeman, B. Liu, Z. Xin, H. Zhao, M. D. Serby,
bulk which may further influence the membrane insertion
energies. This is an interesting result because this indicates that
P. R. Kym, T. S. Suhar, H. T. Smith, N. Cao, R. Yang, R. S. Janis,
the kinetics of the membrane partitioning process of lipidated
proteins can be regulated by changing the saturation state of the
acyl chains, along with other molecular parameters such as the
charge density of the hypervariable region of the Ras protein.
To conclude, we have developed a chemical probe that enables
the accurate and systematic identification of S-bound fatty acids in
their native state. A spiked internal standard allows the relative
quantification of the fatty acids. The analysis of the S-acylome in
different cell lines as well as in an enriched N-Ras revealed that
J. A. Krauser, S. P. Cepa, D. W. Beno, H. L. Sham, C. A. Collins,
T. K. Surowy and H. S. Camp, J. Med. Chem., 2007, 50, 3086.
14 O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner,
M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer and
P. I. H. Bastiaens, Science, 2005, 307, 1746.
15 O. W. Lindwasser and M. D. Resh, Proc. Natl. Acad. Sci. U. S. A., 2002,
99, 13037.
16 O. Rocks, M. Gerauer, N. Vartak, S. Koch, Z. P. Huang, M. Pechlivanis,
J. Kuhlmann, L. Brunsveld, A. Chandra, B. Ellinger, H. Waldmann and
P. I. H. Bastiaens, Cell, 2010, 141, 458.
17 K. Weise, G. Triola, L. Brunsveld, H. Waldmann and R. Winter,
J. Am. Chem. Soc., 2009, 131, 1557.
proteins are modified with fatty acids of various chain lengths and 18 Y. X. Chen, S. Koch, K. Uhlenbrock, K. Weise, D. Das, L. Gremer,
L. Brunsveld, A. Wittinghofer, R. Winter, G. Triola and H. Waldmann,
structures. These previously overlooked heterogenous acyl chains
Angew. Chem., Int. Ed., 2010, 49, 6090.
may have an additional role in regulating protein localization and
19 K. Weise, S. Kapoor, C. Denter, J. Nikolaus, N. Opitz, S. Koch,
function. Thus, AFM studies and FRET-based kinetics assays
indicated that the unsaturated N-Ras presents an increased ten-
dency toward clustering and higher insertion kinetic rate constants
G. Triola, A. Herrmann, H. Waldmann and R. Winter, J. Am. Chem.
Soc., 2011, 133, 880.
20 H. Cao, K. Gerhold, J. R. Mayers, M. M. Wiest, S. M. Watkins and
G. S. Hotamisligil, Cell, 2008, 134, 933.
compared to that of its saturated counterpart. Unfortunately, our 21 A. Vogel, G. Reuther, K. Weise, G. Triola, J. Nikolaus, K. T. Tan,
current method as well as direct LC-MS/MS analysis25 or metabolic
C. Nowak, A. Herrmann, H. Waldmann, R. Winter and D. Huster,
Angew. Chem., Int. Ed., 2009, 48, 8784.
22 C. Nicolini, J. Baranski, S. Schlummer, J. Palomo, M. Lumbierres-
labelling,4,7,26 still require overexpression and purification of indivi-
dual proteins. Alternative hydroxylamine derivatives will be explored
in the future to overcome this limitation. All in all, we are convinced
that the combined use of hydroxylamine derivatives and HRMS-
Burgues, M. Kahms, J. Kuhlmann, S. Sanchez, E. Gratton,
H. Waldmann and R. Winter, J. Am. Chem. Soc., 2006, 128, 192.
23 M. Dwivedi, T. Mejuch, H. Waldmann and R. Winter, Angew. Chem.,
Int. Ed., 2017, 56, 10511.
based analysis may strongly contribute to a better understanding 24 S. Kapoor, A. Werkmuller, R. S. Goody, H. Waldmann and R. Winter,
J. Am. Chem. Soc., 2013, 135, 6149.
25 E. Thinon, J. P. Fernandez, H. Molina and H. C. Hang, J. Proteome
of the biochemistry of S-acylated proteins.
This work was supported by the Max Planck Society (Max
Res., 2018, 17, 1907.
Planck Partner Group to G. T.) with partial support from the 26 E. Thinon, A. Percher and H. C. Hang, ChemBioChem, 2016, 17, 1800.
Chem. Commun.
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