Difference between revisions of "Team:Bielefeld-CeBiTec/Project/toolbox/photolysis"

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            <h2> Photolysis </h2>
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<div class="content"><h3> Short summary  </h3>
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                  The non-canonical amino acid (ncAA) 2-nitrophenylalanine (2-NPA) has the special property to induce a cleavage of the peptide backbone when irradiated with light of a wavelength of 365 nm. To demonstrate the usage of 2-NPA, we designed a fusion protein of the green fluorescent protein (GFP) and streptavidin connected by a glycine-glycine-serine-linker. The streptavidin compound will form a stable and highly specific non-covalent bond to biotin, so that the fusion protein can easily be immobilized on any biotinylated surface. The GFP is used as a fluorescence tag so that the fusion protein can easily be identified through its fluorescent properties. The immobilized fusion protein can then be irradiated with light to induce the cleavage of the peptide backbone and elute the target protein
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                <h3> Photolysis of peptide chains </h3>
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                    This system is an alternative procedure of site-specific protein cleavage compared to a cleavage by proteases or chemicals. A big advantage of this light-induced cleavage is, that it can be used almost universally on any purpose. It prevents disadvantages like an undesired cleavage of the target protein by undetected cleavage sides or a denaturation of the protein through the reagents when cleaved chemically. The system has a wide range of possible applications, such as inactivating proteins by cleaving them, activating them by cleaving an inactive pre-protein releasing an active form, or a combination with other methods as demonstrated in our light-induced elution.
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                <h3> Explanation of the ncAA </h3>
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                    The ncAA 2-NPA is able to photochemically cleave the polypeptide backbone by an cinnoline forming reaction based on the photochemistry of (2-nitrophenyl)ethane derivatives which are used as photochemical caging groups. Peters et al. (2009) report that upon photolysis, the nitrobenzyl group rearranges to the α-hydroxy-substituted nitrosophenyl group. The nitroso group then undergoes an additional reaction with the N-terminal amide group to generate the cyclic azo product. Subsequent hydrolysis of the activated carbonyl group affords the terminal cinnoline and carboxylate products [3].
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f1/T--Bielefeld-CeBiTec--photo_cleavage_mechanism.jpg">
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<p class="figure subtitle"><b>Figure 1: Proposed mechanism of photocleavage reaction by Peters et al [3].</b>
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                <h3> Characteristics of the ncAA </h3>
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                        <li> Name: 2-Nitro-L-phenylalanine
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                        <li> Short: 2-NPA
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                        <li> CAS: 19883-75-1
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                        <li> MW: 210.19
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                        <li> Storage:          2-8°C
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                        <li> Source: apolloscientific
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                        <li> Prize: 5g - £205.00
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                        <li> Function:          induces a cleavage of the peptide backbone when radiated with ʎ=365nm
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Revision as of 01:03, 27 August 2017

Photolysis

Short summary

The non-canonical amino acid (ncAA) 2-nitrophenylalanine (2-NPA) has the special property to induce a cleavage of the peptide backbone when irradiated with light of a wavelength of 365 nm. To demonstrate the usage of 2-NPA, we designed a fusion protein of the green fluorescent protein (GFP) and streptavidin connected by a glycine-glycine-serine-linker. The streptavidin compound will form a stable and highly specific non-covalent bond to biotin, so that the fusion protein can easily be immobilized on any biotinylated surface. The GFP is used as a fluorescence tag so that the fusion protein can easily be identified through its fluorescent properties. The immobilized fusion protein can then be irradiated with light to induce the cleavage of the peptide backbone and elute the target protein

Photolysis of peptide chains

This system is an alternative procedure of site-specific protein cleavage compared to a cleavage by proteases or chemicals. A big advantage of this light-induced cleavage is, that it can be used almost universally on any purpose. It prevents disadvantages like an undesired cleavage of the target protein by undetected cleavage sides or a denaturation of the protein through the reagents when cleaved chemically. The system has a wide range of possible applications, such as inactivating proteins by cleaving them, activating them by cleaving an inactive pre-protein releasing an active form, or a combination with other methods as demonstrated in our light-induced elution.

Explanation of the ncAA

The ncAA 2-NPA is able to photochemically cleave the polypeptide backbone by an cinnoline forming reaction based on the photochemistry of (2-nitrophenyl)ethane derivatives which are used as photochemical caging groups. Peters et al. (2009) report that upon photolysis, the nitrobenzyl group rearranges to the α-hydroxy-substituted nitrosophenyl group. The nitroso group then undergoes an additional reaction with the N-terminal amide group to generate the cyclic azo product. Subsequent hydrolysis of the activated carbonyl group affords the terminal cinnoline and carboxylate products [3].

Figure 1: Proposed mechanism of photocleavage reaction by Peters et al [3].

Characteristics of the ncAA

  • Name: 2-Nitro-L-phenylalanine
  • Short: 2-NPA
  • CAS: 19883-75-1
  • MW: 210.19
  • Storage: 2-8°C
  • Source: apolloscientific
  • Prize: 5g - £205.00
  • Function: induces a cleavage of the peptide backbone when radiated with ʎ=365nm