N-terminal Cys

Note The eytoehrome subunit is membrane-assoeiated via a diaeylglyeerol moiety on its N-terminal Cys residue ... 

Intein cleavage and release of expressed protein with N-terminal Cys... 

The following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

The peptide segment to be ligated to the polymer supported bound peptide was dissolved in 6M Gn-Cl, 0.1 M Na3P04 (pH 7.0), 0.1 M Met, and 1% PhSH (10-50 mM peptide), added to the N-terminal Cys-peptide-resin, and allowed to react at rt. Ligation reactions were complete in 2-24 h. 

It is possible that N-terminal Cys would react toward periodate in the same way, but this seems not to have been tested in practice. 

Fig. 3.14. Farnesylation at the C-terminus. The signal sequence for farnesylation is the C-termi-nal sequence CAAX. In the first step a farnesyl moiety is transferred to the cystein in the CAAX sequence. The farnesyl donor is farnesyl pyrophosphate and the responsible enzyme is farnesyl transferase. Subsequently, the three C-terminal amino acids are cleaved (A alanine, X any amino acid) and the carboxyl group of the N-terminal Cys-residue becomes methylated.

Figure 9 RP-HPLC Analysis of the Dimerization Behavior of c-Myc and Max Leucine Zippers Containing a N-Terminal Cys-Gly-Gly Linker (a) Equal Amounts of Reduced c-Myc and Max before Air Oxidation, (b) after 48 h of Air Oxidation, (c) before Equilibrium Redox Experiment, and (d) after Equilibrium Redox Experiment (after 24h)l61laJ ...

In this method, the cysteine-thioester cyclization generates a cyclic peptide 86a (see Scheme 23) with a Xaa-Cys bond whose thiol moiety is then used for tethering to the core through an S-alkylation reaction.191 The requirement for the cyclization reaction is a linear precursor 84 containing both an N-terminal Cys and a C-terminal thioester. Such a peptide precursor can be conveniently synthesized by a stepwise solid-phase synthesis on a thioester resin 81 using Boc chemistry (Scheme 22). Cleavage by HF after assembly of the peptide sequence will produce the desired precursor with an N-terminal Cys and a C-terminal thioester 84. The crude peptide is then purified by RP-HPLC and the purified unprotected peptide is then circularized in aqueous conditions buffered at pH > 7.0. 

In this method, the cyclic peptide is generated through cysteine-perthioester cyclization to afford a Xaa-Cys bond whose thiol is then used for the second ligation step to the core through a thiol addition reaction (Scheme 25).[S71 For the perthioester cyclization, the linear precursor 91 consists of an activated N-terminal Cys(Npys) and a C-terminal thiocarboxylic acid which favors cyclization due to intramolecular mixed disulfide formation between them. 

PNA with the desired N-terminal Cys or bromoacetyl functionalities may be purchased from Panagene (Korea). Alternatively, PNA synthesis may be achieved using an APEX 396 Robotic Peptide Synthesizer. Following final deprotection, the PNA is analyzed and purified using HPLC, and the molecular mass verified by MALDI-TOF mass spectrometry. 

Figure 19-9. LC/ESI-MS/MS product ion mass spectra of the doubly charged ions of the N-terminal tryptic peptide Ti of (a) IFN-a-2b m/z 658) and (b) Iso-4 (m/z 693). N-terminal Cys-Asp was identified as the modified fragment.

Hackenberger CP, Chen MM, Imperiali B. Expression of N-terminal Cys-protein fragments using an intein refolding strategy. Bioorg. Med. Chem. 2006 14(14) 5043-5048. 

Fig. 2 Structural model of Mycobacterium tuberculosis recA mini intein (PDB 2IMZ) after the splicing reaction. The N-terminal Cys is in close proximity of the C-terminal succinimide...

As exemplified above, a ligation needs an N-terminal Cys and a C-terminal thioester as prerequisites. In chemical synthesis these two conditions are obviously easily achieved. However, recombinant proteins are synthesized with an N-terminal Met and the C-terminus is just a free carboxyl group. There are various methods available to generate an N-terminal Cys. In a commonly used strategy a specific protease cleaving site is introduced. The Tobacco Etch Virus (TEV) protease recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves the... 

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