Engineering encodable lanthanide-binding tags into loop regions of proteins.

Area of Science:

• Biophysics
• Structural Biology
• Protein Engineering

Background:

• Lanthanide-binding tags (LBTs) are crucial for studying protein structure, function, and dynamics using techniques like NMR spectroscopy and X-ray crystallography.
• Interleukin-1β (IL1β) is a model protein frequently studied using biophysical methods.

Purpose of the Study:

• To investigate the utility of LBTs inserted into loop regions of IL1β for biophysical studies.
• To assess the impact of LBT insertion on protein structure, function, and binding interactions.

Main Methods:

• Insertion of LBTs into three loop positions (L, R, S) of IL1β with varying spacer lengths (1-3).
• Luminescence studies for Tb3+ binding affinity.
• X-ray crystallography and NMR spectroscopy (1H−15N HSQC) to assess structural integrity.
• In vitro binding assays to evaluate native partner interactions.
• NMR spectroscopy (RDCs, paramagnetic shifts) for structural analysis and modeling.

Main Results:

• All nine LBT-IL1β constructs exhibited tight Tb3+ binding (low nanomolar range).
• LBT insertion did not significantly alter IL1β structure or its binding to its native partner.
• X-ray crystallography phasing was achieved using only lanthanide signals.
• Residual dipolar couplings (RDCs) indicated rigid incorporation of LBTs, particularly the LBT-2 series.
• Paramagnetic NMR data allowed for structural modeling of the IL1β-R2 construct.

Conclusions:

• Encodable LBTs are versatile biophysical tags for proteins, especially when inserted into loop regions.
• LBTs facilitate structural and dynamic studies via NMR and crystallography without compromising protein function.
• This approach is applicable to proteins with known structures or those predicted via homology modeling.