Backbone double-mutant cycle analysis quantifies hydrogen-bond energies in proteins

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Protein structure relies on noncovalent interactions, many of which are not fully understood.
• Quantifying backbone interactions experimentally is challenging due to difficulties in obtaining reliable energy measurements.
• Previous efforts have been complicated by confounding factors, hindering integration into structural models.

Purpose of the Study:

• To adapt double-mutant cycle analysis for quantifying protein backbone interactions.
• To provide the first experimental quantification of weak, intra-strand hydrogen bonds.
• To develop a reliable method for probing backbone interactions in proteins.

Main Methods:

• Adapted double-mutant cycle analysis combined with chemical peptide synthesis.
• Selectively probed backbone interactions, minimizing confounding factors.
• Validated the approach by quantifying canonical, cross-strand hydrogen bonds in beta-sheet proteins.

Main Results:

• The adapted method accurately quantified canonical, cross-strand hydrogen bonds, agreeing with previous findings.
• Provided the first experimental quantification of weak, intra-strand hydrogen bonds.
• Found individual intra-strand hydrogen bonds contribute approximately 0.2 kcal/mol, significant due to their frequency.

Conclusions:

• The developed approach offers a reliable method for experimentally quantifying protein backbone interactions.
• This technique can be applied to various protein systems, aiding structural and folding research.
• Experimental quantification of intra-strand hydrogen bonds supports their role in protein folding and misfolding.