Tryptophanyl contributions to apomyoglobin fluorescence resolved by site-directed mutagenesis

Area of Science:

• Biochemistry
• Protein Folding
• Spectroscopy

Background:

• Sperm-whale apomyoglobin contains two tryptophanyl residues (W7 and W14) whose fluorescence properties are sensitive to their microenvironment.
• Understanding protein conformational changes is crucial for elucidating protein function and dysfunction.
• Previous studies suggested a charge-transfer complex involving W14 and histidine, influencing fluorescence.

Purpose of the Study:

• To resolve the individual emission properties of W7 and W14 in sperm-whale apomyoglobin.
• To investigate the impact of acid and guanidine denaturation on apomyoglobin conformation and tryptophanyl fluorescence.
• To re-evaluate the cause of fluorescence changes observed during acid-induced transitions.

Main Methods:

• Site-directed mutagenesis was used to create W7F and W14F apomyoglobin mutants.
• Fluorescence spectroscopy was employed to monitor changes in tryptophanyl residue emission.
• Apomyoglobin and its mutants were subjected to denaturation by varying pH (acid) and guanidine concentration.

Main Results:

• Acid denaturation (pH 6.0 to 4.0) caused minor microenvironmental changes affecting both W7 and W14, not a charge-transfer complex.
• Acidic conditions led to increased fluorescence yield and a red shift for W7 due to K79 release.
• Guanidine denaturation increased W14 fluorescence in an intermediate state, while W7 fluorescence remained constant.

Conclusions:

• The fluorescence decrease at pH 6.0 is attributed to subtle structural alterations, not a charge-transfer complex.
• Acidic pH induces a partly folded state affecting W7's fluorescence via K79 interaction.
• Unfolding pathways of apomyoglobin may differ based on the denaturant used, highlighting distinct conformational responses.