CHAPTER 1S
The Perception of Hydrophobic Clusters in the Native
and Partially Unfolded States of a Protein
G. VANDERHEEREN 1 and I. HANSSENS 1
1
Introduction
1.1
Hydrophobic Interactions as the Driving Force in Protein Folding
The folding mechanism of globular proteins is dominated by hydrophobic interactions. They are believed to playa main role in the formation of a-helices and
~-structures and also in the further folding into a partially folded protein with
fluctuating tertiary structure (Miranker and Dobson 1996). This fluctuating protein is called a 'molten globule'. The final transition to the native structure with
tight close-packed contacts between the amino acid side-chains is governed by
short-range electrostatic interactions (Levitt et al. 1997). The residues in the
hydrophobic core of the partially (un)folded protein are accessible to external
agents and can effect binding interactions with hydrophobic probes, such as 1,1'bis( 4-anilino-5-naphthalenesulfonate) (bis-ANS). As the fluorescence quantum
yield of those molecules greatly increases upon binding to hydrophobic sites, this
interaction with molten globule-like intermediates of the protein leads to a large
fluorescence increase. This property has been used to estimate the population of
the molten globule state in protein folding studies (Ptitsyn et al. 1990; Teschke et
al. 1993; Das and Surewicz 1995). The use of these hydrophobic probes for the
characterization of the molten globule state has been questioned by other
research groups since they are suspected of inducing changes in the native protein conformation. In this way Coco and Lecomte (1994) revealed perturbations
in the structure of native-like apomyoglobin upon ANS-binding. Shi et al. (1994)
observed that bis-ANS, which preferentially binds to the molten globule state of
DnaK, shifts the equilibrium from the native to the molten globule state. Engelhard and Evans (1995) observed that ANS, used in a kinetic study of the alactalbumin refolding process, stabilizes the dye-bound intermediates.
With this contribution we demonstrate that bis-ANS is helpful in obtaining
information on the degeneration of the hydrophobic domains at different stages
of the thermal unfolding of a-lactalbumin. Furthermore we discuss the interaction of the hydrophobic probe with the native protein state of a-lactalbumin and
I Interdisciplinary Research Center, Kathalieke Universiteit Leuven, Campus Kartrijk, B-8500
Kartrijk, Belgium.
The Perception of Hydrophobic Clusters in the Native
and Partially Unfolded States of a Protein
G. VANDERHEEREN 1 and I. HANSSENS 1
1
Introduction
1.1
Hydrophobic Interactions as the Driving Force in Protein Folding
The folding mechanism of globular proteins is dominated by hydrophobic interactions. They are believed to playa main role in the formation of a-helices and
~-structures and also in the further folding into a partially folded protein with
fluctuating tertiary structure (Miranker and Dobson 1996). This fluctuating protein is called a 'molten globule'. The final transition to the native structure with
tight close-packed contacts between the amino acid side-chains is governed by
short-range electrostatic interactions (Levitt et al. 1997). The residues in the
hydrophobic core of the partially (un)folded protein are accessible to external
agents and can effect binding interactions with hydrophobic probes, such as 1,1'bis( 4-anilino-5-naphthalenesulfonate) (bis-ANS). As the fluorescence quantum
yield of those molecules greatly increases upon binding to hydrophobic sites, this
interaction with molten globule-like intermediates of the protein leads to a large
fluorescence increase. This property has been used to estimate the population of
the molten globule state in protein folding studies (Ptitsyn et al. 1990; Teschke et
al. 1993; Das and Surewicz 1995). The use of these hydrophobic probes for the
characterization of the molten globule state has been questioned by other
research groups since they are suspected of inducing changes in the native protein conformation. In this way Coco and Lecomte (1994) revealed perturbations
in the structure of native-like apomyoglobin upon ANS-binding. Shi et al. (1994)
observed that bis-ANS, which preferentially binds to the molten globule state of
DnaK, shifts the equilibrium from the native to the molten globule state. Engelhard and Evans (1995) observed that ANS, used in a kinetic study of the alactalbumin refolding process, stabilizes the dye-bound intermediates.
With this contribution we demonstrate that bis-ANS is helpful in obtaining
information on the degeneration of the hydrophobic domains at different stages
of the thermal unfolding of a-lactalbumin. Furthermore we discuss the interaction of the hydrophobic probe with the native protein state of a-lactalbumin and
I Interdisciplinary Research Center, Kathalieke Universiteit Leuven, Campus Kartrijk, B-8500
Kartrijk, Belgium.