Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Proteins exhibit local structural fluctuations (LSFs), also known as breathing motions, which are vital for their function but remain poorly understood.
• LSFs were previously defined by amide hydrogen exchange (HX) experiments, identifying opening events that expose backbone amides.

Purpose of the Study:

• To characterize LSFs in myoglobin (Mb) using size-dependent thiol-disulfide exchange (SX).
• To complement HX data by investigating motions that affect side chain packing.
• To determine the size and properties of structural opening events using probes of varying sizes.

Main Methods:

• Utilized single cysteine-containing myoglobin variants.
• Employed size-dependent thiol-disulfide exchange (SX) with thiosulfonate reagents (126-274 Da).
• Analyzed the relationship between the free energy of opening and the radius of gyration of probe reagents.

Main Results:

• SX provided insights into motions disrupting side chain packing.
• The free energy of opening increased linearly with probe reagent size for buried cysteine residues.
• The slope parameter varied over 10-fold across different cysteine positions, indicating substantial variation in breathing motion sizes.

Conclusions:

• LSFs vary considerably in size across different locations within a protein.
• SX is a valuable technique for probing protein flexibility and the nature of LSFs.
• This study offers insights into the rigidity and flexibility of proteins in their native states.