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Dual
Polarisation Interferometry (DPI) offers a truly quantitative analytical
technique, rather than a simple ‘mass sensor’ response, providing absolute
density measurements and structural dimensions of immobilised proteins at high
resolution (1).
Dimethyl Sulphoxide
- DMSO
The use of combinatorial and parallel
chemistry has made high-throughput screening the major tool for discovery of
pharmaceutically active compounds. Large collections of low molecular weight
organic molecules (< 500 Da) represent a primary source of discovery
chemistry for bioscreening. Dimethyl sulphoxide (DMSO) is an important solvent
for small molecule studies as it provides a nearly universal approach for the
solubilisation of small molecules. Because of its physicochemical properties,
high solvent power, low chemical reactivity and relatively low toxicity, DMSO
has become the solvent of choice for sample storage and handling in the
pharmaceutical industry, particularly in the initial stages of high-throughput
screening where storage of candidate molecules in DMSO is routine.
The Effects of
DMSO
The effects of DMSO on protein structure and
function are extremely varied. When used as a co-solvent at high concentration
DMSO has great potential to give misleading results in bioavailability
measurements, by acting as a permeation enhancer, a denaturant, or even an
inhibitor (2). Additionally, DMSO has a much higher refractive index (RI) than
common buffer solutions, which can make it difficult to produce reliable
readings from DMSO-containing samples using angular-based optical biosensor
technology such as SPR. The combination of density changes and DMSO effects on
the protein has made experimental design very arduous and interpretation of
results rather complex. This is compounded by the limited dynamic range
first-generation biosensors have for high RI solvents such as DMSO, making some
experiments simply unworkable.
DMSO in Drug
Discovery
In the pharmaceutical industry, knowledge of
the three-dimensional structure of a specific protein target facilitates the
structurally informed drug-discovery process. This application note explores the
effects that DMSO as a co-solvent has on lysozyme structure through
conformational changes induced upon exposure to a concentration gradient of
aqueous DMSO solutions. This work also demonstrates the wide dynamic range of DPI
and its capability for analysis even when using high RI buffer solutions,
without the need for complicated pre-calibration procedures.
Experimental
DPI
experiments were performed on a Farfield
AnaLight® instrument. This method assumes that the AnaLight® fluidic system has been cleaned according to the
recommended procedures (Farfield Technical Note 002) and that the injection needle and
syringes are free of contaminants before use. The surface used in these studies
was an amine functionalised silicon oxynitride chip activated with
bis(sulphosuccinimidyl)suberate (BS3). The temperature of the samples was
controlled throughout to 20°C. Water used in buffer preparation was deionised
and free from organic impurities. All buffers and reagents were analytical grade
or higher, and solutions were degassed prior to use.
Calibration
The amine-functionalized chip was calibrated
using 80% ethanol and water (Farfield Technical Note 001). DMSO was prepared as a
co-solvent in PBS (10mM phosphate, 150mM NaCl, pH7.4) at 1%, 2%, 5%, 10% and 20%
(v/v) and then flowed over the untreated chip at 50µl/min. As a control, similar
concentrations of H2O in PBS were injected over the chip surface at
the same flow rate.
Immobilisation of
Lysozyme
After activating the surface with BS3 linker
(3mg/ml in PBS), lysozyme (2mg/ml) prepared in PBS was introduced into the
buffer flow and allowed to covalently couple to the chip, via NHS-ester reactive
groups and primary amines on the protein, for 6 minutes at 30µl/min. To block
any unreacted linker or surface groups, an injection of ethanolamine (0.1M in
PBS) was passed over the chip (Farfield Technical Note 003).
DMSO–Lysozyme
Interactions
After stabilising the immobilised lysozyme
with sufficient rinsing, the protein layer was exposed alternately to PBS and
increasing concentrations of DMSO (1%, 2%, 5%, 10% and 20% (v/v)) in PBS for 4
minutes per cycle at a flow rate of 50µl/min. A similar experiment was performed
in which H2O was substituted for DMSO in PBS at the same
concentrations.
Results and
Discussion
Immobilisation of
Lysozyme
Table 1 shows the physical properties of
lysozyme covalently immobilised to an amine functionalised chip surface prior to
DMSO treatment. Protein size and molecular ‘footprint’ values are in close
correlation with crystallographic dimensions of 45Å (long axis) and 30Å (short
axis) and 900Å2 respectively, suggesting a non-constrained
conformation of lysozyme post-immobilisation.
Table 1. Structural properties of covalently coupled lysozyme protein,
immobilised to an amine functionalised surface activated with BS3 NHS-ester
reactive groups
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|
|
|
|
|
|
Lysozyme |
1.457 |
40.2 |
2.692 |
882 |
DMSO-Lysozyme
Interactions
Whilst looking for the diminutive signals
associated with molecular reorientation, effects of differences in the buffer,
particularly density, need to be accounted for if the measurement is to be
quantified. This is simply and unambiguously achieved with the
AnaLight® instrument by subjecting the untreated chip surface to a series of
trial solutions before immobilisation of protein. Bulk density differences are
corrected by subtracting values for initial injections of DMSO over the
untreated chip from the corresponding immobilised lysozyme data. Conformational
changes within the protein structure induced by exposure to the different
solvent regimes may then be accurately measured. Figure 1 shows concentration
dependent effects of DMSO on lysozyme protein, and molecular reorientation upon
returning to PBS.
Figure 1. Dimensional (blue) and density (green) changes indicating
conformational rearrangement of lysozyme during exposure to a range of DMSO
concentrations and upon returning to PBS running buffer
Changes in
the Lysozyme Structure
Clear changes are measured in the lysozyme
structure each time the solvent is switched. Measured size of the lysozyme
molecule decreases when it is subjected to DMSO-containing buffer compared to
PBS alone. Density increases correspondingly, particularly at higher
concentrations of DMSO. In neat DMSO the structure of lysozyme is known to
approach a random coil with complete lack of tertiary structure (3), a process
that appears to be initiated in the lysozyme well before 100% DMSO, arguably at
concentrations used in this study. However, the exaggerated compression of the
protein structure caused by higher DMSO concentrations eventually leads to a
progressive and irreversible reordering of the lysozyme structure on returning
to PBS, as shown by the increasing protein size from left to right in Figure 1.
Data in Figure 1 also demonstrates the ability of the
AnaLight® to resolve structures in high RI solvents such as 20% DMSO in PBS
(measured by DPI to have a RI of 1.3667). In fact, the upper limit for the
RI of a solvent that may be successfully resolved in DPI
experiments is approximately 1.50, a value higher than that achievable using
other optical techniques such as SPR.
Correcting for Salt Concentration Effects
A parallel experiment was performed where
water, rather than DMSO, was added to PBS in order to correct for salt
concentration effects. Changes in pH and salt concentration can alter
electrostatic interactions between charged amino acids in a protein structure.
Increasing salt concentration (by reducing the concentration of H2O
in the sample) reduces the strength of ionic binding by providing competing ions
for the charged residues, as pairing of salt ions with charged groups of the
protein shields intra-molecular repulsion. Measured size of the lysozyme
molecule increases when it is subjected to water-containing buffer compared to
PBS alone. Initial structural dimensions are spontaneously resumed each time the
protein is returned to PBS buffer (data not shown), suggesting that these
structural processes are completely reversible.
This confirms that DMSO-containing buffer is
having a structural effect on the protein that is distinct from salt
concentration effects.
Conclusions and
Benefits
AnaLight® DPI systems offer next-generation technology,
providing valuable insights into the relationship between protein structure and
function under different buffer regimes, eg. pH, salt concentration and organic
buffer constituents. ‘Biosensor’ techniques such as SPR and QCM cannot reveal
this level of quantitative structural information. This application note clearly
demonstrates that DPI is a powerful technology with the sensitivity and dynamic
range to give reliable data on protein structural changes even in high RI
background solutions. DPI
has also revealed that DMSO can cause irreversible conformational changes in
target proteins. Whilst structural effects are seen in equivalent concentrations
of water, changes cause by water, are completely reversible. It is likely that
DMSO has an effect on the structural integrity, and therefore function, of a
wide range of proteins. The implications of these structural changes in
therapeutic target proteins should be carefully considered during
high-throughput screening.
These experiments show DPI
can bring a new level of understanding to the assessment of protein structure by
giving real time information on immobilized protein dimensions, density, surface
coverage and orientation. AnaLight® gives the researcher a unique combination of
high-resolution data in real time on thickness, density and surface coverage
from a bench top technique. AnaLight® is an important enabling tool for biophysicists,
giving them the ability to:
·
Connect functional and structural events
in a single set of high-content measurements, in real time.
·
Measure the structural changes in proteins
that result from solvent effects.
·
Qualify false positives in high-throughput
screening that are caused by solvent effects
·
Design experiments in which the solvent
regime is ideal for the target protein rather than being limited by instrument
capabilities
References
1.
G.Cross, A Reeves, S Brand, J Popplewell,
L Peel, M Swann & N Freeman. Biosens. Bioelectron. 19 (2003)
383-390.
2.
H Johannesson, V Denisov & B
Halle. Protein Science
6 (1997) 1756-1763.
3.
T Knubovets, J Osterhout & A Kilanov.
Biotech. Bioeng. 63 (1999) 242-248. | 
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Farfield Group
