Real-time protein unfolding: A method for determining the kinetics of
native protein denaturation using a quantitative real-time thermocycler
Kyle K. Biggar*, Neal J. Dawson & Kenneth B. Storey
Department of Biology, Carleton University, Ottawa, ON, Canada
ABSTRACT
The stability of a protein can be monitored by many different techniques. However,
these protocols are often lengthy, consume large amounts of protein, and require
expensive and specialize instruments. In this study, we present a new protocol to analyze
the kinetics of protein unfolding utilizing a quantitative real-time thermocycler. By
using modified qRT-PCR equipment, it is possible to analyze the effects of a wide range
of denaturants (and their interactions with temperature change) on protein stability on
a microplate platform where multiple samples can be run in parallel under virtually
identical conditions and with highly sensitive detection. Using this set-up, researchers
can determine the concentration of denaturant that results in a half-maximal rate of
protein denaturation (KND), the maximum rate of denaturation (Dmax), and the
cooperativity of individual denaturants in protein unfolding (µ-coefficient). In the
present study we use hexokinase as a model protein and urea as a model denaturant to
illustrate this new method and the kinetics of protein unfolding that it can supply. This
method allows the researcher to explore a large number of denaturants, at either
constant or variable temperatures, within the same assay. Overall, this method provides
an estimate of denaturation kinetics that was previously inaccessible.
INTRODUCTION
The limits of survival for many living organisms are marked by extreme environmental
conditions: for example, 0 to 12.5 for pH and approximately -18 to 121°C for temperature.
While some organisms that live in extreme environments can evade the stress, other
organisms must adapt their intracellular molecules for survival. To maintain proper
functioning of their intracellular proteins, these organisms display structural adaptations
that influence both protein stability and activity. Consequently, there is great interest in
understanding the mechanisms involved in the stabilization of protein structure.
EQUIPMENT DESIGN & SET-UP
Figure 1. The dual emission and detection
channels of the MyiQ2 system offer an
affordable option for detecting common green
fluorescent dyes, such as SYBR Green I (qRTPCR), and Sypro Orange (Protein melt).
This methods work by utilizing a modified
qRT-PCR thermocycler to monitor protein
unfolding in the presence of a hydrophobic
fluorescent dye (typically SYPRO orange). The
typical analysis conducted is to determine the
rate of change in fluorescence intensity over
time as a function of various concentrations of
denaturants/stabilizers. This method is able to
obtain novel real-time kinetic analysis of
protein unfolding. By combining the use of (I)
a 96-well PCR microplate, (II) small sample
volumes (20 µl), (III) a short procedure time (50
min), and (IV) the ability to monitor
denaturation at various temperatures (4.0 –
100.0°C), this protocol reduces both time and
technical variation.
SYPRO-ORANGE DYE
As a protein unfolds, it exposes
hydrophobic residues that are able to
interact with SYPRO orange dye,
resulting in an increase in fluorescent
emission that can be detected by qRTPCR optics. In the present study we used
hexokinase (HK) as a model protein and
urea as a model denaturant. To
determine the effect of urea concentration
on the rates of protein unfolding, protein
samples were incubated at various urea
levels in the presence of SYPRO orange
dye. At each urea concentration, the
relative rate of unfolding of the protein
was recorded as an increase in the
fluorescence of SYPRO orange over time.
MATERIALS & METHODS
Protein Denaturation Kinetics
Experimental Set-up
SYPRO orange (Invitrogen, Cat# S-6650) can be
monitored in quantitative RT-PCR instruments
using the filters commonly provided with the
machines: FAM (485 nm) and ROX (625 nm) for
excitation and emission, respectively (Figure 1).
For the present experiments, we modified a
BioRad MyIQ2 thermocycler (BioRad, Cat# 1709790), installing the appropriate filters into a
separate channel than the one utilized for SYBRgreen based experiments.
Protein Preparation
Hexokinase (HK) purified from the yeast,
Saccharomyces cerevisiae (Sigma Aldrich, Cat#
H-5500), was diluted to a final concentration of
0.2 mg/ml in a phosphate buffer (100 mM
potassium phosphate, pH 7.0, 150 mM NaCl).
The SYPRO orange dye was diluted to a 40X
stock in potassium phosphate buffer and used
from this concentration for all experiments.
Protein incubations with urea concentrations
ranging from 0 to 4.5 M in potassium phosphate
buffer. Each sample consisted of 2.5 μL of 0.2
mg/ml purified HK, 2.5 μL of 40X SYPRO
orange dye and 15 μL of potassium phosphate
buffer (various urea concentrations) for a final
volume of 20 μL. Fluorescent measurements were
initiated immediately. Fluorescent reads were
taken every 5 seconds on a temperature gradient
consisting of 5°C, 15°C, 25°C, and 35°C for 50
minutes. The change in fluorescence was plotted
using a simple scatter plot, and the linear
portions of the curve were truncated and
analyzed using a linear regression.
The resulting slopes were plotted on a second graph
using Kinetics v.3.5.1 program and analyzed using the Hill
equation. From this, KND, Dmax and µ-coefficient values
were calculated. Respectively, these values were defined as
the concentration of denaturant that resulted in a halfmaximal rate of native protein denaturation (KND), the
calculated maximum rate of protein denaturation (Dmax),
and the extent of denaturant cooperativity (µ-coefficient).
PROTEIN MELTING KINETICS
120
PULSE PROTEOLYSIS - VALIDATION
ENZYME ACTIVITY- VALIDATION
Figure 7. Effect of urea on HK protein unfolding. Relative
amount of folded HK remaining after 50 min incubation with
urea as assessed following thermolysin treatment and SDSPAGE detection. Data are means ± SEM (n = 4 trials).
Figure 8. Effect of urea on maximum HK activity. Mean HK
activity after 50 min incubation with various concentrations
of urea. Data are means ± SEM (n = 4 trials).
DISCUSSION
To determine the rate of HK denaturation in urea, each resulting slope was then plotted
against the concentration of urea. These values gave a sigmoidal plot that was analyzed
using the Hill equation. The following results where determined using this method:
RESULTS
RAW FLUORESCENT DATA
RESULTS
Dma
x
100
KND
Figure 3. Representative raw data collection from a
quantitative real-time thermocycler. An increase in
fluorescence results from an increase in protein unfolding,
and incorporation of SYPRO orange to protein structure.
Note: Each cycle has a pre-defined time component (5 sec).
Figure 5. Effect of urea on velocity of HK denaturation.
Result of urea incubation on the maximum velocity of HK
protein denaturation as determined by the slope of relative
fluorescence over time at various concentrations of urea in
potassium phosphate buffer.
PROTEIN MELTING
TEMPERATURE EFFECTS ON KND
• The KND at 25°C was determined to be 3.31 ± 0.22 M (n = 4) (Fig. 5).
• A Dmax of 120.67 ± 0.88 ΔF.U./min was calculated for HK denaturation by urea at 25°C
(Fig. 5).
• This measurement of cooperativity, defined herein as the ‘µ-coefficient’, for the influence
of urea on HK denaturation was calculated to be 5.63 ± 0.61 (Fig. 5).
• No significant influence of temperature on KND was seen between 5°C and 15°C (p<0.05)
(Fig. 6).
• At higher temperatures, 25°C and 35°C, the KND for urea significantly decreased (Fig. 6).
Here we have presented a new technique to quantify the kinetics of protein denaturation.
We obtained a half-maximal rate of native protein denaturation (KND), maximum rate of
denaturation (Dmax), and extent of denaturant cooperativity (µ-coefficient) under several
experimental conditions. Our method is very simple, requires only small sample volumes,
and provides large amounts of reproducible real-time data in a relatively short experimental
protocol. The method allows the researcher to quickly and efficiently determine the
susceptibility of protein structure to many different denaturants while also allowing the
variable of temperature change to be used as a means to further manipulate denaturation.
Stability data could also be collected as a function of competing stabilizing agents. This
method provides an opportunity for researchers to explore protein stability with readily
available equipment, and allows current studies that evaluate protein stability to explore
changes in degradation kinetics.
FUTURE STUDIES
This study has made contributions to studying protein unfolding in an inexpensive and simple method.
Using Real-time monitoring it may be possible to expand the resolution of the existing protocol to
study multiple-state unfolding. Increasing the frequency of fluorescent reads and decreasing the rate
of unfolding (lowered temperature) may allow for enough resolution to study multiple-state unfolding
in some proteins.
Figure 2. Representation of the excitation (solid) and
emission (dashed) spectra of SYPRO orange
fluorescent dye in bovine serum albumin. Shaded
areas represent the relative range of light transmitted
through the BioRad 485 ± 30 nm excitation and 625 ±
30 nm emission filters used in this experiment.
Figure 4. Initial protein denaturation monitored by
fluorescence. Protein denaturation assessed via relative
changes in fluorescence units (F.U.) due to SYPRO orange
binding over the initial seconds of exposure to a denaturant.
Figure 6. Effect of urea on the KND of HK protein
denaturation at various temperatures. Data are means ± SEM
(n = 4 trials). ‘a’ – significantly different from the 5°C value,
‘b’ –significantly different from 15°C, ‘c’ – significantly
different from 25°C, (p<0.05).
ACKNOWLEDGEMENTS
This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of
Canada. K.B.S. holds the Canada Research Chair in Molecular Physiology, K.K.B. held an NSERC CGSD postgraduate
fellowship.