Surface Engineering of Top7 to Facilitate Structure Determination.

Area of Science:

• Protein engineering
• Structural biology
• Biochemistry

Background:

• Top7 is a de novo designed protein lacking evolutionary traces, making it ideal for studying intrinsic protein properties and for protein engineering.
• Initial attempts at X-ray structure determination of Top7 were hindered by weak diffraction.

Purpose of the Study:

• To facilitate crystal structure determination of the de novo protein Top7.
• To engineer Top7 variants with improved crystallographic properties for structural analysis.
• To obtain a high-resolution structure of an engineered Top7 variant for future applications.

Main Methods:

• Surface residue mutations (Top7sm1, Top7sm2) were introduced to enhance crystallization.
• X-ray crystallography was employed to determine protein structures.
• Intermolecular interface mutations (Top7sm2-I68R) were designed to alter crystal packing.

Main Results:

• Surface mutants Top7sm1 and Top7sm2 showed improved crystallization and diffraction to ~1.7 Å.
• Despite improved diffraction, high R values prevented structure finalization for surface mutants.
• The mutant Top7sm2-I68R, with altered intermolecular interactions, diffracted to 1.4 Å, enabling structure determination (R/Rfree = 0.20/0.24).

Conclusions:

• Surface mutations can facilitate crystal packing and improve diffraction resolution.
• Engineered Top7 variants, particularly Top7sm2-I68R, offer convenient structure determination.
• Top7sm2-I68R serves as a valuable model protein for structural biology and protein engineering research.