Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates

• Published in: Protein science Vol. 8; no. 5; pp. 1099 - 1103
• Main Authors: D'SILVA, PATRICK R., LALA, ANIL K.
• Format: Journal Article
• Language: English
• Published: Bristol Cambridge University Press 01-05-1999; Cold Spring Harbor Laboratory Press
• Subjects: Animals; bovine α‐lactalbumin; Cattle; Chemistry, Physical - methods; Cyanogen Bromide - pharmacology; diazofluorene; Diazonium Compounds - pharmacology; Fluorenes - pharmacology; hydrophobic photolabeling; Lactalbumin - chemistry; Models, Molecular; molten globule state; Photolysis; Protein Folding; protein folding intermediates; protein folding intermediates; molten globule state; diazofluorene; bovine α-lactalbumin; hydrophobic photolabeling
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.8.5.1099

Recent advances in attempts to unravel the protein
folding mechanism have indicated the need to identify the
folding intermediates. Despite their transient nature,
in a number of cases it has been possible to detect and
characterize some of the equilibrium intermediates, for
example, the molten globule (MG) state. The key features
of the MG state are retention of substantial secondary
structure of the native state, considerable loss of tertiary
structure leading to increased hydrophobic exposure, and
a compact structure. NMR, circular dichroism, and fluorescence
spectroscopies have been most useful in characterizing
such intermediates. We report here a new method for structural
characterization of the MG state that involves probing
the exposed hydrophobic sites with a hydrophobic photoactivable
reagent—2[3H]diazofluorene.
This carbene-based reagent binds to hydrophobic sites,
and on photolysis covalently attaches itself to the neighboring
amino acid side chains. The reagent photolabels α-lactalbumin
as a function of pH (3–7.4), the labeling at neutral
pH being negligible and maximal at pH 3. Chemical and proteolytic
fragmentation of the photolabeled protein followed by peptide
sequencing permitted identification of the labeled residues.
The results obtained indicate that the sequence corresponding
to B (23–34) and C (86–98) helix of the native
structure are extensively labeled. The small β-domain
(40–50) is poorly labeled, Val42 being the only residue
that is significantly labeled. Our data, like NMR data,
indicate that in the MG state of α-lactalbumin, the
α-domain has a greater degree of persistent structure
than the β-domain. However, unlike the NMR method,
the photolabeling method is not limited by the size of
the protein and can provide information on several new
residues, for example, Leu115. The current method using
DAF thus allows identification of stable and hydrophobic
exposed regions in folding intermediates as the reagent
binds and on photolysis covalently links to these regions.