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Edlich, Christian; Muhle‐Goll, Claudia

doi:10.1023/a:1025493410292

Cited by 2 publications

(2 citation statements)

References 9 publications

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“…This contained well-dispersed peaks of uniform line width as expected for a small rigid globular protein. The chemical shifts of the isolated Z1 were similar to those previously reported for Z1 within the Ig -Ig pair Z1-Z2 (Edlich and Muhle-Goll, 2003). The [ 1 H, 15 N]-HSQC spectrum of Z1 FLAG (Fig.…”

Section: Localization Of Structural Alterations In Z1 Flag By Nmrsupporting

confidence: 84%

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The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition

Bruning

Barsukov

Franke

et al. 2012

Protein Engineering Design and Selection

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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.

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Section: Localization Of Structural Alterations In Z1 Flag By Nmrsupporting

confidence: 84%

“…Initial values for the chemical shifts were obtained from the Z1-Z2 tandem (BioMagResBank, entry 5760; Edlich and Muhle-Goll, 2003), and adjusted for Z1. Due to the large differences between the [ 1 H, 15 N]-HSQC of Z1 and Z1 FLAG , the resonances of Z1 FLAG were assigned independently using the 3D spectra.…”

Section: Nuclear Magnetic Resonance Spectroscopymentioning

confidence: 99%

The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition

Bruning

Barsukov

Franke

et al. 2012

Protein Engineering Design and Selection

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The Ig Doublet Z1Z2: A Model System for the Hybrid Analysis of Conformational Dynamics in Ig Tandems from Titin

et al. 2006

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Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

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Cited by 2 publications

References 9 publications

The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition

The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition

The Ig Doublet Z1Z2: A Model System for the Hybrid Analysis of Conformational Dynamics in Ig Tandems from Titin

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