Communications
which indicates that the refolded Rheb lipoprotein and the
recombinant Rheb protein adopt a similar secondary struc-
ture.
To determine the functionality of the semisynthetic Rheb
lipoprotein, we monitored its binding to an effector that
inhibits the dissociation of nucleotides from the Ras-like
G protein. This interaction can be followed in a real-time
fluorescence-based guanine nucleotide dissociation inhibition
[
15]
(
GDI) assay, which indicates a conformational switch of the
G protein between different nucleotide-loaded states. Semi-
synthetic Rheb lipoprotein was fully loaded with fluorescently
labeled methylanthraniloyl (mant) nucleotides (i.e. mant-
GppNHp, a nonhydrolysable GTP analogue, or the analogous
mant-GDP). Upon the addition of excess unlabeled nucleo-
tides (GppNHp or GDP), the bound mant nucleotides are
exchanged for unlabeled nucleotides. Since the fluorescence
intensity of the Rheb-bound mant nucleotide is approxi-
mately twice as high as the fluorescence intensity of the free
mant nucleotide, the nucleotide-exchange reaction can be
monitored as decay of the fluorescence signal. Recombinant
forms of possible Rheb effectors, such as mTOR itself or the
mTORC1 complex, are not known. However, we recently
developed a mutated form of the Ras binding domain of the
Raf protein (Raf-RBD) that specifically recognizes GTP-
bound Rheb and therefore inhibits the dissociation of the
[
16]
nucleotide from active Rheb. We applied both wild-type
and mutated Raf-RBD proteins to active (mant-GppNHp-
loaded) or inactive (mant-GDP-loaded) semisynthetic Rheb
lipoprotein in the GDI assay. In the presence of mutated Raf-
RBD, the nucleotide exchange of mant-GppNHp-bound
Rheb was slowed down, whereas in the presence of wild-
type Raf-RBD, there was no change relative to the intrinsic
rate (Figure 2b). In contrast, for mant-GDP-bound Rheb,
neither mutated nor wild-type Raf-RBD affected the rate of
the nucleotide-exchange reaction. These results prove that
the semisynthetic Rheb lipoprotein was correctly folded and
functional, as indicated by conformational switching between
the active and the inactive state.
Scheme 2. Synthesis of the farnesylated and carboxymethylated Rheb
protein 1. a) Fmoc-Ser-OAll (8), pyridine, DMF/CH Cl ; b) [Pd(PPh ) ],
The K-Ras4B protein 4 was synthesized by a similar
strategy (Scheme 1b). K-Ras4B also does not contain an
extra cysteine residue at its C terminus for protein ligation. In
this case, a cysteine residue was inserted at the N-terminal end
of the polybasic amino acid sequence adjacent to the
farnesylated C-terminal cysteine methyl ester. The resulting
dodecapeptide 5, which features a total of eight lysine
residues, was synthesized on a solid support (see Scheme S1
in the Supporting Information). The growing peptide chain
was anchored to a chlorotrityl linker through the side chain of
the lysine residue nearest the C terminus. The other lysine
side chains were protected with the allyloxycarbonyl (Alloc)
2
2
3 4
PhSiH , CH Cl ; c) H-Cys(Far)-OMe (10), HATU, collidine, DMF/
3
2
2
CH Cl ; d) 1) piperidine in DMF; 2) Fmoc-AA-OH, HCTU, DIPEA,
2
2
DMF; AA=Ser(Trt), Lys(Mtt), Gly; repeat; e) 1) piperidine in DMF;
) Fmoc-AA-OH, PyBOP, DIPEA, DMF; AA=Gln, Ser(Trt); repeat;
2
f) 1) piperidine in DMF; 2) Fmoc-Cys(StBu)-OH, PyBOP, collidine,
DMF/CH Cl ; g) TFA, TES, CH Cl ; 29% overall yield from 11;
2
2
2
2
h) TCEP, ligation buffer; i) RhebD11-MESNA thioester 3, Tris–HCl
buffer, pH 7.4, MESNA, CTAB, 20% yield. All=allyl, DIPEA=N,N’-
diisopropylethylamine, DMF=dimethylformamide, Far=farnesyl,
Fmoc=9-fluorenylmethoxycarbonyl, HATU=2-(7-aza-1H-benzotriazol-
-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HCTU=(2-(6-
1
chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluoro-
phosphate), PyBOP=(benzotriazol-1-yloxy)trispyrrolidinophosphonium
hexafluorophosphate, TES=triethylsilane, TFA=trifluoroacetic acid,
Tris=2-amino-2-hydroxymethylpropane-1,3-diol.
[17]
group to enable their orthogonal palladium(0)-catalyzed
deprotection in the presence of the other functionalities in the
peptide. After cleavage from the solid support, the peptide
could be precipitated readily and was obtained in 15% overall
yield after preparative HPLC purification. Similarly to the
Rheb thioester 3, the truncated K-Ras4B protein core
residues of Rheb are highly flexible and do not determine the
[
12]
overall secondary structure,
we used the recombinant
RhebD11-MESNA thioester 3 as a reference for comparison
with the structure of the refolded Rheb lipoprotein 1. The CD
spectra of both proteins superimpose very well (Figure 2a),
thioester 6 was generated through expression in the
IMPACT vector pTWIN2 (New England Biolabs), purifica-
tion on a chitin-affinity column, and release after treatment
6
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 6090 –6095