Exploring the equilibrium unfolding and the
kinetics of goat α-lactalbumin under different
experimental conditions
Allel Chedad
Results
Figure 4 shows the time-course of refolding of GLA in the absence (panel A) and presence of 10 mM Ca2+
(panel B) measured by the CD ellipticity change at 222 nm, which is characteristic for the α-helical content
during refolding. The ellipticity change recorded in the far-UV region, indicates that in the absence or in
the presence of Ca2+, 52 and 66 % of the total signal occurs within the dead-time of the measurement (2
ms). These results indicate that an unresolved folding event accumulates within the dead-time of the
measurement. The refolding rate has increased with a factor 600 in Ca2+-GLA compared to the rate
constant measured for apo-GLA.
• Geboren op 27 september 1974 te Zwevegem.
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A
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[θ]222 (deg.cm2.dmol-1)
2
-1
[θ]222 (deg.cm .dmol )
• Kandidaturen Natuurkunde aan de K.U.Leuven Campus Kortrijk (1993-1995).
• Licenties Natuurkunde K.U.Leuven (1995-1997).
• Wetenschappelijke medewerker aan het Labo voor Agrarische Bouwkunde KU.Leuven (April
1998- September 2001).
• Assistent Natuurkunde aan de K.U.Leuven Campus Kortrijk (Oktober 2001- Juni 2006).
• Doctoraatsverdediging op 21 juni 2006.
• Promotor: Prof. Dr. Herman Van Dael.
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U
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Goat α-lactalbumin (GLA) is a small globular protein consisting of 123 amino acids with a molecular
weight of 14 kDa. The protein consists of two structural domains: a large α-helical domain (1-39 and
85-123) and a shorter β-sheet domain (40-84). Like all other lactalbumin, GLA possesses a strong
Ca2+-binding site situated at the interface of the two domains.
A remarkable feature of α-LA is its ability to populate an equilibrium partially folded state, which is
also known as the molten globule state (MG), under easily accessible experimental conditions such as
acidic pH, depletion of Ca2+-ions or moderate concentrations of denaturant (Arai and Kuwajima,
2000). The MG-state is characterised by (1) the presence of a pronounced amount of secondary
structure, (2) the absence of most of the native tertiary interactions, and (3) the presence of a loosely
packed hydrophobic core (Kuwajima, 1996).
• Thermal unfolding curves can be constructed by measuring the λmax of Trp-emission as a function of
temperature under equilibrium conditions.
Ca2+-binding site
N
0
50
100
150
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0.0
200
0.2
0.4
0.6
time (s)
0.8
1.0
Figure 4: Time course of the refolding of apo-GLA (A) and holo-GLA (B) monitored by the CD ellipticity at 222 nm at
25°C. The refolding reaction is initiated by an 11 fold dilution of the unfolded protein in 6M GdnHCl. The curves are
well fitted to a single exponential. In panel (A), the rate constant and amplitude of the phase are k= 0.03 s-1 and A=1287
deg·cm2·dmol-1, respectively; and in panel (B) k=20.8 s-1 and A=1978 deg·cm2·dmol-1. The arrows in both panels indicate
the signal level of the unfolded protein in refolding conditions. Buffer conditions are 20 mM Tris, 80 mM Na+, 2 mM
EDTA at pH 7.5 for the apo-form (panel A) and 10 mM Ca2+ for the holo-form (panel B).
φ-value analysis
In this work we report on how a single Trp to Phe mutation (W118F, W104F, W60F and W26F) affects the
stability of the kinetic intermediate, the transition and the native state, respectively. The most commonly
used method to interpret the results of protein engineering studies on folding kinetics is by φ-value
analysis. The objective of this analysis is to evaluate the importance of a mutated residue in stabilising the
folding intermediate and transition state structure. A φ-value of 0 means that at the site of mutation the
structure is unfolded as much as it is in the denatured state. A φ-value of 1 means that at the site of
mutation the structure is folded as much as it is in the native state (Figure 5).
Table 1: The φ-values calculated for the kinetic intermediate
and the transition state of the four Trp-mutants.
C
A-helix
W118
W104
W26
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time (s)
Introduction
B
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wt-GLA
W118F
W104F
W60F
W26F
φI
-
- 0.06
0.95
0.90
0.94
φ‡
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- 0.07
0.96
0.84
0.93
B-helix
W60
Figure 5: Schematic representation of the effect of
mutation on the free energy of the native and the
transition state.
C-helix
D-helix
Figure 1: Crystal structure of GLA generated from coordinates deposited in the Brookhaven Protein
Data Bank, 1HFY (Pike et al., 1996). The side chains of the four Trp residues (W118, W104, W60 and
W26) are represented by sticks.
The main goal of this work is to characterise partially folded states of GLA populated at equilibrium
and in the kinetics under a variety of experimental conditions (Ca2+, pH, Na2SO4 and Trp-mutation).
Studying the equilibrium unfolding and the folding and unfolding kinetics of a protein under different
experimental conditions is a powerful approach for revealing the mechanism by which the protein
attains its native and functional state.
Experimental set-up
Many physical properties of biological molecules can be inferred from the interaction with
electromagnetic radiation. Due to the asymmetric nature of the peptide bond and some side-chains,
proteins are optically active, in that they interact differently with left and right circularly polarised
light. When circularly polarised light passes through a sample the left and right components can be
differentially absorbed. This results in the circularly polarised light becoming elliptically polarised,
and the phenomenon is called circular dichroism. Within this work, near- and far-UV circular
dichroism and fluorescence spectroscopy are used as techniques to monitor changes in secondary and
tertiary structure during kinetic and equilibrium maesurements of GLA.
The φ-values of W26F, W60F and W104F are close to 1 which means that, when Trp26 or Trp60 or Trp104
is replaced by Phe, the kinetic intermediate and the native state are destabilized by the same amount. In
other words in these proteins the intermediate state at the mutation site has a native character. In contrast,
the φ-value for the intermediate of the W118F is 0, reflecting that in the intermediate state the mutation site
is still unfolded. Similar results were found for φ‡ indicating that the mutation of Trp118 has no specific
effect on the transition state.
Effect of Na2SO4 on the folding pathway of GLA
Our data demonstrate that the addition of Na2SO4 clearly stabilises the native, intermediate and transition
state for GLA where the accumulation of the intermediate is unfavourable. It further demonstrate that the
addition of Na2SO4 increases the α-helical content of the burst-pahse intermediate under conditions where
its accumulation is unfavourable. This suggests that the local interactions and thus the formation of
secondary structure is predominant for stabilising the kinetic intermediate. As a result, the formation of
secondary structure precedes the formation of the native-like interactions. Our data can be interpreted
within the framework of a hierarchical model. The φ-values for the Trp mutants further support our
conclusion based on the kinetic analysis. For all the Trp-mutants, the φ-values are as high in the transition
state as in the kinetic intermediate, suggesting that their interactions are consolidated with progress towards
the native state. These findings are thus in full agreement with a hierarchical model for folding which is
represented schematically in Figure 6.
Apo-GLA
kobs
slow
2+
Ca2+Ca -GLA
Ca2+
kobs
fast
Figure 2: principle of circular dichroism.
Stopped-flow is a technique for following the kinetics of a reaction in solution, usually in the millesecond time range. Two syringes rapidly inject volumes of reactant solutions through a mixing
chamber towards the optical cell (Figure 3). The flow of the mixed solution through the optical cell is
then suddenly stopped when the receiving syringe (stop syringe) is completely filled and a trigger
initiates the acquirement of the data. After mixing, the reaction is followed as a function of time by
some spectroscopic technique with a rapid response such as fluorescence, circular dichroism or
absorption. We used the stopped-flow technique for monitoring the folding and unfolding reactions of
GLA.
Stop syringe and Trigger
Refolding buffer
Detector (fluorescence, CD or
absorption)
Protein in 6M denaturant
unfolded
Ram
Figure 3: Schematic representation of the stopped-flow technique
t < 2ms
MG-state
t > 2ms
Native state
Figure 6: The folding pathway of GLA. Folding begins with structure that are local in sequence and limited in stability and
docks further into the native tertiary structure of the protein after populating a MG-state. Experiments carried out in the
presence of Ca2+ show a 600-fold increase in the refolding rate (Figure 4).
Conclusions
• The refolding rate has increased with a factor 600 in holo-GLA compared to the rate constant measured for
apo-GLA.
• Our data suggest that GLA folds according to a hierarchical mechanism.
• A wide range of experimental conditions must be tested before explaining the folding mechanism of a
protein.
Fluorescence intensity (a.u.)
Optical cell
t << 2ms
Formation of secondary structure
with marginal stability
References
Arai M. and Kuwajima K. (2000). Role of the Molten Globule State in Protein Folding. Advances in Protein Chemistry 53, 209-282.
Kuwajima K. (1996). The Molten Globule State of α-lactalbumin. Faseb Journal 10, 102-109.
Time (s)
Pike A.C.W., Brew K. and Acharya K.R. (1996). Crystal structures of guinea-pig, goat and bovine α-lactalbumin highlight the enhanced conformational
flexibility of regions that are significant for its action in lactose synthase. Structure 4, 691-703.