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claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Mills BJ, Mu Q, Krause ME, Laurence JS - Bioconjug. Chem. (2014)

Bottom Line: This approach has been much more effective with large lanthanide series metals than smaller transition metals.The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag.The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The University of Kansas , Lawrence, Kansas 66045, United States.

ABSTRACT
Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

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Illustration of the potential location of Ni-claMP within the EGF molecule. (a) Schematic of EGF molecule; residuesaffected by addition of the claMP Tag on the C-terminusare shown in gray. (b) Electrostatic surface map of EGF with the Ni-claMP Tag added onto the C-terminus. The negatively chargedNi-claMP complex is hypothesized to associate withthe positively charged pocket shown in blue.
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fig6: Illustration of the potential location of Ni-claMP within the EGF molecule. (a) Schematic of EGF molecule; residuesaffected by addition of the claMP Tag on the C-terminusare shown in gray. (b) Electrostatic surface map of EGF with the Ni-claMP Tag added onto the C-terminus. The negatively chargedNi-claMP complex is hypothesized to associate withthe positively charged pocket shown in blue.

Mentions: The three disulfide bonds presentin EGF are the primary contributingfactors to the protein structure. As such, conservation of the nativedisulfide bonds is essential to preserve protein structure and maintainbiological function. Formation of the native disulfide bonds in EGFoccurs through multiple iterations of incorrect cysteine pairs untilthe correct disulfide network is achieved,29 and addition of the claMP Tag introduces two additionalnon-native cysteine residues into EGF, which could interfere withthe native disulfide network. To validate that addition of the claMP Tag to EGF did not affect protein structure, two-dimensionalheteronuclear NMR was used to compare the variants to native EGF (Figure 5).27 A 1H–15N HSQC spectrum provides a fingerprint of the protein; eachamide from the backbone generates one peak, which reflects the uniqueconformation of the corresponding residue within the structured protein. 1H–15N HSQC spectra obtained for EGF andEGF-Ni-claMP are highly similar and have specific,localized differences. The spectra show that the native fold of theprotein is maintained in the presence of the claMPTag, as the majority of the residues in both spectra overlay. Thedisulfide network is also maintained; the chemical shift positionsof the five assigned cysteine residues remain unperturbed. The datashow that the tertiary structure is not altered by the addition ofthe claMP Tag. In the EGF-Ni-claMP spectrum, a few notable differences from native EGF are observed;residues near the C-terminus of the protein (K48, W49/50, L52, andR53) are shifted. There also are differences in peak intensity observedbetween the variants for M21, G36, and L47. Because NMR is exquisitelysensitive to small changes in local environment, the presence of themetal-bound claMP Tag would be expected to influencethe chemical shift of neighboring residues. The chemical shift positionsof EGF with and without the tag were compared and the differencesmapped onto the structure of EGF to show the residues affected bythe tag. The structure reveals that the negatively charged Ni-claMP module likely associates with a neighboring patchof positive charge (Figure 6).


claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Mills BJ, Mu Q, Krause ME, Laurence JS - Bioconjug. Chem. (2014)

Illustration of the potential location of Ni-claMP within the EGF molecule. (a) Schematic of EGF molecule; residuesaffected by addition of the claMP Tag on the C-terminusare shown in gray. (b) Electrostatic surface map of EGF with the Ni-claMP Tag added onto the C-terminus. The negatively chargedNi-claMP complex is hypothesized to associate withthe positively charged pocket shown in blue.
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fig6: Illustration of the potential location of Ni-claMP within the EGF molecule. (a) Schematic of EGF molecule; residuesaffected by addition of the claMP Tag on the C-terminusare shown in gray. (b) Electrostatic surface map of EGF with the Ni-claMP Tag added onto the C-terminus. The negatively chargedNi-claMP complex is hypothesized to associate withthe positively charged pocket shown in blue.
Mentions: The three disulfide bonds presentin EGF are the primary contributingfactors to the protein structure. As such, conservation of the nativedisulfide bonds is essential to preserve protein structure and maintainbiological function. Formation of the native disulfide bonds in EGFoccurs through multiple iterations of incorrect cysteine pairs untilthe correct disulfide network is achieved,29 and addition of the claMP Tag introduces two additionalnon-native cysteine residues into EGF, which could interfere withthe native disulfide network. To validate that addition of the claMP Tag to EGF did not affect protein structure, two-dimensionalheteronuclear NMR was used to compare the variants to native EGF (Figure 5).27 A 1H–15N HSQC spectrum provides a fingerprint of the protein; eachamide from the backbone generates one peak, which reflects the uniqueconformation of the corresponding residue within the structured protein. 1H–15N HSQC spectra obtained for EGF andEGF-Ni-claMP are highly similar and have specific,localized differences. The spectra show that the native fold of theprotein is maintained in the presence of the claMPTag, as the majority of the residues in both spectra overlay. Thedisulfide network is also maintained; the chemical shift positionsof the five assigned cysteine residues remain unperturbed. The datashow that the tertiary structure is not altered by the addition ofthe claMP Tag. In the EGF-Ni-claMP spectrum, a few notable differences from native EGF are observed;residues near the C-terminus of the protein (K48, W49/50, L52, andR53) are shifted. There also are differences in peak intensity observedbetween the variants for M21, G36, and L47. Because NMR is exquisitelysensitive to small changes in local environment, the presence of themetal-bound claMP Tag would be expected to influencethe chemical shift of neighboring residues. The chemical shift positionsof EGF with and without the tag were compared and the differencesmapped onto the structure of EGF to show the residues affected bythe tag. The structure reveals that the negatively charged Ni-claMP module likely associates with a neighboring patchof positive charge (Figure 6).

Bottom Line: This approach has been much more effective with large lanthanide series metals than smaller transition metals.The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag.The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The University of Kansas , Lawrence, Kansas 66045, United States.

ABSTRACT
Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

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