[Show abstract][Hide abstract] ABSTRACT: Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with
robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the
characterization of Ig domains of intracellular origin—in particular, modular components of the titin myofilament. These Ig
belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its
CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras
using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate
that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the
domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal
titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold,
interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the
intracellular Ig scaffold for targeted functionalization.
Preview · Article · Feb 2012 · Protein Engineering Design and Selection
[Show abstract][Hide abstract] ABSTRACT: The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.