Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1

Becker, Christian F. W.; Hunter, Christie L.; Seidel, R.; Kent, Stephen B. H.; Goody, Roger S.; Engelhard, Martin

doi:10.1073/pnas.0831227100

Cited by 56 publications

(27 citation statements)

References 27 publications

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“…A wide range of chemical modifications can now be introduced with the precise control that total chemical synthesis allows (23,(27)(28). For example, fluorescent unnatural amino acids can be introduced (29)(30)(31), allowing a more direct, and so more quantitative, fluorescence-imaging approach than the biotin-streptavidin Alexa Fluor 488 protocol used here. Fluorescently labeled analogs will allow a variety of fluorescence resonance energy transfer (FRET) studies to be performed, and may also allow the simultaneous recording of single-channel conductance and single-molecule FRET.…”

Section: Discussionmentioning

confidence: 99%

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Clayton

Shapovalov

Maurer

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed. R ecently, ion channel research has been aided by the discovery of bacterial ion channels that share many key features of mammalian channels (1-3). Some of these bacterial channel proteins are smaller and more amenable to expression and purification than mammalian channels. Importantly, bacterial channels can often be reconstituted into vesicles and other synthetic membrane systems, allowing detailed functional characterization (4-6). Furthermore, several bacterial channels have been described at atomic resolution by x-ray crystallography (7-11), providing opportunities for correlating structure and function in a key class of membrane protein.Although the preparation of many medium-sized watersoluble proteins by chemical ligation is now a routine procedure (12)(13)(14), the synthesis of ion channels and other membrane proteins presents unique challenges. Many hydrophobic peptides have sequences that couple inefficiently, meaning that the synthesis of each segment must be optimized. Hydrophobic peptides also have limited solubility and tend to aggregate. Furthermore, after synthesis is complete the native oligomeric protein must be formed from unfolded monomers. However, recent advances in chemical ligation methodologies have permitted the semisynthesis (15) and the total chemical synthesis of membrane proteins, including the 97-residue type III (single membrane-spanning segment) M2 protein of the influenza virus (16). The synthetic M2 protein assembled into the native tetrameric form on reconstitution into dodecylphosphocholine micelles.Here, we describe the chemical synthesis of two polytopic membrane proteins, the mechanosensitive ion channels from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (TbMscL), and the vesicle reconstitution of these channels into a form functionally similar to that of the recombinant protein.Multimilligram quantities of Ec-MscL and Tb-MscL were generated by optimizing synthesis, purification, and ligation protocols previously developed fo...

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Section: Discussionmentioning

confidence: 99%

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Clayton

Shapovalov

Maurer

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…To test this, we then examined the effect of Man-A on Ras activity using the Ras RBD assay (Ras Binding Domain of Raf assay), which only pulls down active Ras:GTP. 34 Untreated WEHI-231 cells were found to contain constitutive Ras activity (Fig. 1d).…”

Section: Effects Of Manumycin-a On Ras Effector Status Ras and Protementioning

confidence: 99%

Reactive oxygen species‐dependent destruction of MEK and Akt in Manumycin stimulated death of lymphoid tumor and myeloma cell lines

Sears

Daino

Carey

2008

Intl Journal of Cancer

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Manumycin-A (Man-A) is a farnesyltransferase inhibitor (FTI), which was originally identified as an effective tumoricide against several cancers, especially ones harboring constitutively active Ras. However, it is becoming apparent that Man-A can stimulate tumor death independently of FTases. Antioxidant treatment blocked Man-A-stimulated DNA damage and reversed Man-Ainhibited tumor growth. However, the precise molecular details of how these reactive oxygen species (ROS) influence cell signaling modules are poorly understood. We examined how ROS may modulate death and survival pathways in a panel of tumor cells. Man-A treatment resulted in a massive induction of superoxide anion (AEO 2 2 ) only in Man-A-sensitive tumors. Within 1 hr, Man-A caused the ROS-dependent activation of caspases 9 and 3. In this time-frame, the Ras-Raf target, MEK, and the survival protein Akt were dephosphorylated in ROS-dependent fashions and then cleaved in ROS and caspase-dependent manners. Pretreatment with ROS scavengers blocked the adverse effects of Man-A, including the processing of caspases and the cleavage of MEK and Akt. These events were noted before any losses in Ras activity or changes in its maturation could be detected. Finally, transfection with cDNAs encoding the antioxidant enzymes catalase, superoxide dismutase and thioredoxin reductase inhibited superoxide induction and apoptosis. Together, our data suggest that the elimination of tumors by Man-A can be independent of the inhibiting of Ras. However, one universal feature observed is the generation of death-triggering intracellular oxidants that appear to directly participate in the select targeting of growth and survival proteins that then either augment or ensure tumor cell death. ' 2007 Wiley-Liss, Inc.Key words: Manumycin; farnesyltransferase inhibitor; MEK; Akt; reactive oxygen species; cleavage; caspase; Ras Ras is a small, 21 kDa, guanosine triphosphate (GTP)-binding protein, which controls numerous cellular functions including growth and proliferation as well as providing scaffolding for other signaling enzymes. Ras regulates the PI3K/Akt and PI3K/mTOR/ p70 S6K signaling modules, which drive cell proliferation, cell growth and survival. 1 Furthermore, the canonical Ras signaling network (Ras/Raf/MEK/ERK) regulates anabolism, induces growth-enhancing genes through transcription factors such as Elk-1 and it also suppresses the activities of proapoptotic proteins. 2 Hence, it is not surprising that 30% of known human tumors and 90% of pancreatic carcinomas, one of the most aggressive and morbid types of cancer, harbor mutated or dysfunctional Ras. [3][4][5] Together these features make Ras an attractive target for cancer therapy.Ras maturation requires the post-translational transfer of a farnesyl hydrocarbon group to its C-terminal cysteine in a process termed prenylation, 6-8 which is essential for the subsequent localization of Ras at the plasma membrane where receptor signals regulate its on and off state. 9 Because prenylation is also required for subsequen...

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“…Precise control over many different chemical modifications will allow direct insight into substrate binding, the catalytic mechanism of DAGK, its oligomeric assembly, and the role of specific amino acid side chains. [30][31][32][33] To this end, we prepared a first DAGK analogue containing a fluorescent probe (7-nitrobenz-2-oxa-1,3-diazole, NBD) linked to the side chain of l-diaminopropionic acid that replaces a native tryptophan residue in position 25 of DAGK and an N-terminal biotin moiety. This variant exhibits a similar k cat value (7 AE 2 s À1 ) to that measured for synthetic DAGK1-121 (see Figure S11 in the Supporting Information).…”

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confidence: 99%

Total Chemical Synthesis of an Integral Membrane Enzyme: Diacylglycerol Kinase from Escherichia coli

et al. 2011

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Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1

Cited by 56 publications

References 27 publications

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Reactive oxygen species‐dependent destruction of MEK and Akt in Manumycin stimulated death of lymphoid tumor and myeloma cell lines

Total Chemical Synthesis of an Integral Membrane Enzyme: Diacylglycerol Kinase from Escherichia coli

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