Part:BBa_K2868014
HHTC-Re + linker
This is a version of the peptide computationally designed by Kozisek et al. (Chemistry, 2008) to bind to copper (HHTC). This metal binding domain has had amino acids replaced for specificity, and has a flexible GSGGSG linker attached to allow for spacing for proper folding of the constituent domains in any fusion proteins being created.
To test whether we could bind multiple metal ions on the same protein and if this relationship would be monotonically increasing, we designed peptides with one, two, and three copper binding domains in tandem (1xHHTC-Re HNLGMNHVLQGNRPLVTQGC, 2xHHTC-Re HNLGMNHVLQGNRPLVTQGCHNLGMNHVLQGNRPLVTQGC, 3xHHTC-Re HNLGMNHVLQGNRPLVTQGCHNLGMNHVLQGNRPLVTQGCHNLGMNHVLQGNRPLVTQGC; see biobrick page for sequence information). These three peptides were synthesized by Elim Biopharmaceuticals (Hayward, CA), and provided as a lyophilized powder at >98% purity. They were also modified to possess N-terminal acetylation/C-terminal amidation to avoid having a charged peptide [17]. 10 mM MES pH 5.5 and the Pierce BCA assay were used for reconstitution and concentration determination, respectively [18].
To check whether the proteins would retain their respective conformations when added in tandem, we used a tool produced by the Zhang lab (University of Michigan) for de novo protein structure prediction (QUARK) [15]. Based on the structures, we were able to hypothesize that the HHTC-Re domains would each be able to bind copper atoms, even when restricted spatially by ordering them sequentially without spacing.
ITC Analysis: Data, Modeling, and Binding Parameters
The results from the ITC experiment to determine binding parameters for peptides 1x-, 2x-, and 3x-HHTC-Re alongside the predicted structure (QUARK Ab Initio program) follows [2]. This program uses Monte Carlo simulations and knowledge of atomic force fields to construct the most probable structural conformation of a protein from just the linear amino acid sequence.
Figure 3: ITC data for the three HHTC-Re peptides. The binding affinity (Ka) was assessed to determine the strength of the interaction, and the results follow: 1x-HHTC-Re Ka = (1.55 ± 0.21) x 106 M-1; 2x-HHTC-Re Ka = (3.73 ± 0.53) x 105 M-1; 3x-HHTC-Re Ka = (1.50 ± 0.05) x 105 M-1.
The binding affinity values in Figure 3 are largely comparable to one another, and we do see a linear trend between the number of HHTC-Re repeats and injections taken to reach saturation, which means that each peptide can bind a linearly increasing amount of copper proportional to the HHTC-Re repeats. This validates our hypothesis underlying our construct design for our fusion proteins, allowing us to bind multiple metals on the same biomolecule. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |