Differentially labeled flaviviral protease-cofactor complex for NMR spectroscopic applications.

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Flaviviruses like Dengue and Zika have RNA genomes translated into polyproteins.
• The NS2B-NS3 protease-cofactor complex matures viral polyproteins.
• This complex is a therapeutic target due to its unique catalytic mechanism involving a peptide cofactor.

Purpose of the Study:

• To develop a method for differentially labeling the NS2B-NS3 protease-cofactor complex.
• To enable isolated NMR spectroscopic study of the flaviviral cofactor.
• To facilitate understanding of cofactor properties and their role in enzyme activity.

Main Methods:

• Selective NMR labeling of the cofactor within the protease-cofactor complex.
• Utilizing 'on-column unfolding' during Ni-NTA chromatography for enzyme-cofactor separation.
• Refolding the labeled cofactor in the presence of unlabeled enzyme to form a differentially labeled complex.

Main Results:

• Successfully rendered the enzyme invisible in NMR spectra by selective labeling of the cofactor.
• Confirmed the desired cofactor fold within the complex using 1H-15N HSQC spectra.
• Demonstrated a scalable and inexpensive protocol for differential labeling.

Conclusions:

• The developed protocol enables selective NMR studies of the flaviviral protease cofactor.
• This method is valuable for investigating cofactor structure-function relationships.
• The technique is applicable to other enzyme systems involving essential peptide cofactors.