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Circular permutation of T4 lysozyme

T Zhang¹, E Bertelsen, D Benvegnu

¹Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.

|November 23, 1993

Summary

Protein folding is not dictated by the order of chain synthesis. A circularly permuted phage T4 lysozyme variant folds efficiently, retaining structure and activity, demonstrating flexibility in protein architecture.

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Area of Science:

Biochemistry
Structural Biology
Molecular Biology

Background:

Protein folding is crucial for biological function.
The relationship between polypeptide synthesis order and final protein structure is complex.
Phage T4 lysozyme is a well-studied enzyme used as a model system.

Purpose of the Study:

To investigate if the linear order of amino acid sequence dictates protein folding.
To construct and characterize a circularly permuted variant of phage T4 lysozyme.
To assess the impact of circular permutation on protein structure, stability, and enzymatic activity.

Main Methods:

Construction of a circularly permuted T4 lysozyme variant with a six-residue linker.
Enzymatic activity assays to measure cell wall cleavage.
Spectroscopic techniques including circular dichroism, UV absorbance, and fluorescence.
Nuclear magnetic resonance (NMR) spectroscopy for structural analysis.
Reversible denaturation experiments to determine protein stability.

Main Results:

The circularly permuted T4 lysozyme folds efficiently and exhibits normal specific activity.
Spectroscopic analyses reveal minimal changes in protein structure compared to the wild-type.
Denaturation experiments show only a slight reduction (0.8-1.1 kcal/mol) in protein stability.
The permuted protein demonstrates that domain structure is maintained despite altered termini.

Conclusions:

Circular permutation of T4 lysozyme does not significantly disrupt its folding, structure, activity, or stability.
These findings challenge the notion that the sequential order of polypeptide synthesis strictly determines protein fold.
Protein folding is robust and can accommodate significant alterations in chain topology, particularly in multi-domain proteins.

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