Measurement of Conformational Changes in the Structure of

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Measurement of Conformational Changes in the Structure of Transglutaminase on Binding Calcium Ions Using Optical Evanescent Dual Polarisation Interferometry K. Karim, J. D. Taylor, and D. C. Cullen*

Cranfield Health, Cranfield University, Silsoe, Bedfordshire MK45 4DT, United Kingdom M. J. Swann and N. J. Freeman*

Farfield Scientific Ltd., Farfield House, Southmere Court, Electra Way, Crewe Business Park, Crewe, Cheshire CW1 6GU, United Kingdom

The conformational changes occurring when the protein transglutaminase binds calcium ions have been studied using the optical evanescent technique of dual polarization interferometry (DPI) implemented via a dual slab waveguide structure. Immobilized transglutaminase layers of 4-5 nm in thickness were obtained, which when challenged with calcium ions underwent a contraction of ∼0.5 nm (depending on the concentration of calcium) and an increase in refractive index of ∼1 × 10-2. The affinity constant for the calcium binding was found to be in the range of 0.95 ( 0.2 mM. The results reported are in good agreement with those found in the literature obtained by other techniques. It has also been shown that the structural changes occurring during the binding event are considerably larger than the mass changes that take place; thus, DPI offers a potentially valuable method to study real-time structural changes occurring to proteins when they bind metal ions. Here we report a study of the conformational changes occurring in the protein transglutaminase, specifically tissue transglutaminase (tTG) from guinea pig liver, when it binds calcium ions, by the use of a new optical evanescent technique of dual polarization interferometry (DPI) implemented via a dual slab waveguide structure. * To whom correspondence should be addressed. [email protected]; [email protected]. 10.1021/ac051254b CCC: $37.00 Published on Web 03/17/2007

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© 2007 American Chemical Society

Transglutaminases are enzymes involved in the post-translational modification of proteins, forming isopeptide bonds at glutamine residues.1,2 The primary function of these enzymes appears to be in the stabilization of tissues and as such their occurrence is widespread, being found in both extracellular and cellular fluids. There is evidence that transglutaminases may be bifunctional, also having capabilities to perform the role of G-proteins in cell signaling pathways.3,4 Factor XIII is one of the best characterized transglutaminases, being the last enzyme to be activated in the blood coagulation cascade increasing the mechanical strength and chemical stability of blood clots. It is a tetramer consisting of two catalytic “a” subunits and two noncatalytic “b” subunits (which are thought to play a role in stabilizing the a units).5 The protein-modifying activities of transglutaminases play a role in areas such as programmed cell death, cross-linking membrane, and cytoskeletal proteins.6,7 This activity has been (1) Greenberg, C. S.; Birckbichler, P. J.; Rice, R. H. FASEB J. 1991, 5, 30713077. (2) Folk, J. E.; Finnlayson, J. S. Adv. Protein Chem. 1977, 31, 1-133. (3) Nakoaka, H.; Perez, D. M.; Back, K. J.; Das, T.; Husain, A.; Misono, K.; Im, M. J.; Graham, R. M. Science 1994, 264, 1593-1596. (4) Monsonego, A.; Friedmann, I.; Shasni, Y.; Eisenstein, M.; Schwartz, M. J. Mol. Biol. 1998, 282, 713-720. (5) Ichinose, A.; Bottenus, R. E.; Davie, E. W. J. Biol. Chem. 1990, 265, 1341113414. (6) Fesus, L.; Piacentini, M.; Davies, P. J. A. J. Cell Biol. 1991, 56, 170-177. (7) Siefring, G. E.; Apostol, A. B.; Velasco, P. T.; Lorand, L. Biochemistry 1978, 17, 2598-2604.

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shown to be modulated by calcium and GTP.8,9 This modulation is thought to be related to putative conformational changes induced by the modulators. Indirect analysis using shallow-angle neutron scattering, shallow-angle X-ray scattering,10 and circular dichroism spectroscopy11 have suggested that there is a significant increase of 0.8 nm in the gyration radius of the protein on binding calcium, suggesting a significant broadening of the protein structure on binding. Efforts to elucidate conformational changes directly from crystallographic data have not been successful as it is thought that the active form (with calcium bound) is constrained by the crystal lattice.12 tTG is only one of many such protein systems where standard structural determinations are unable to provide the required information. Modern bioscience practices rely heavily on the determination of X-ray crystal structures, but diffraction quality crystals are difficult, sometimes impossible to obtain, and the influence of the crystal lattice on the final structure is a matter of considerable concern. X-ray structures are also “end-point” determinations of structure, the protein being “frozen” in the structure determined from diffraction experiments. As a result of these difficulties, a number of studies have been carried out in which putative structural changes are inferred indirectly from optical or acoustic measurements. While such techniques provide less structural detail, they avoid the need to obtain protein crystals, the presence of a crystal lattice, and provide real-time information, which can provide additional information on the nature of changes taking place. Experiments on optical surface plasmon resonance (SPR) measurements of transglutaminase on the addition of calcium ions have already been reported.13 The transglutaminase used consisted of a single catalytic a subunit, which is considered to bind several calcium ions.14 However, given the relative nature of the measurements, it was not possible to relate these observations directly to data obtained from analytical techniques such as X-ray crystallography and neutron scattering techniques. As the SPR measurements provided a single optogeometric parameter, it was only possible to ascribe a putative structural change to the binding event based on the fact that the responses obtained were at variance with those expected. Multiple-wavelength SPR techniques such as coupled plasmon waveguide resonance (CPWR) have been deployed to look at changes in protein conformation.15 This technique provides structural data on protein conformational changes at resolutions that are comparable with the technique described here. However, CPWR studies have been largely confined to steady-state changes in protein conformation (rather than real time) and have focused on membrane-bound proteins. (8) Achyuthan, K. E.; GreenBurg, C. S. J. Biol. Chem. 1987, 262, 1901-1906. (9) Bergamini, C. M.; Signorini, M.; Caselli, L.; Melandri, P. Biochem. Mol. Biol. Int. 1993, 30, 727-732. (10) Cassadio, R.; Polverini, E.; Mariani, P.; Spinozzi, F.; Carsughi, F.; Fontana, A.; Polverino de Laureto, P.; Matteucci, G.; Bergamini, C. M. Eur. J. Biochem. 1999, 262, 672-679. (11) Di Venere, A.; Rossi, A.; De Matteis, F.; Rosato, N.; Finazzi, A.; Mei, G. J. Biol. Chem. 2000, 275, 3915-3921. (12) Fox, B. A.; Yee, V. C.; Pedersen, L. C.; Le Trongi, I.; Bishop, P. D. J. Biol. Chem. 1999, 274, 4917-4923. (13) Gestwicki, J. E.; Hsieh, H. V.; Pitner, J. B. Anal. Chem. 2001, 73, 57325737. (14) Sigma-Aldrich, Inc.; Product Information Sheet TMG/JRC 9/02. (15) Salamon, Z.; MacLeod, H. A.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 131-152.

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Figure 1. Schematic of the principle and implementation of dual polarization interferometer.

In this paper, we report on a new optical technique, optical evanescent wave dual polarization interferometry, which has been employed to study the immobilization of guinea pig liver tissue transglutaminase to a surface and its subsequent behavior on the introduction of calcium ions and demonstrate the different levels of data analysis that may be undertaken to draw relevant inferences about structural changes occurring in real time. This system was previously studied using a range of analytical techniques enabling comparisons to be made with both other commercial optical biosensors and complimentary analytical techniques such as X-ray crystallography and neutron scattering techniques. THEORY Values of surface adlayer thicknesses and refractive index were determined from real-time measurements of optical phase changes in an optical evanescent dual polarization interferometer comprising an AnaLight Bio200 instrument (Farfield Scientific Ltd., Crewe, UK). The adlayer density and mass can be further derived from these data. The fundamental science underpinning this instrumentation has been described in detail elsewhere.16,17 A brief description is provided below. The measurement technique combines a simple optical waveguide structure and the use of simultaneous and independent optical measurements to obtain analytical information regarding the nature of the immediate environment of the surface of the waveguide structure. Using this approach, it is possible to obtain information on the deposition and characteristics of thin films deposited at the waveguide/liquid interface to subatomic spatial resolution in the dimension orthogonal to the interface. The measurement platform consists of two planar optical waveguides stacked one on top of the other as shown in Figure 1. Polarized light from a laser is fed to the end of the stack, and, therefore, at the moment of entry, the light waves in the top and bottom waveguides are in phase. The top surface of the upper (sensing) waveguide and hence the evanescent wave component of the guided light at the upper waveguide surface/sample interface is exposed to sample. Therefore, any changes in the refractive index experienced by the evanescent wave component of the guided lightsa combination of bulk sample refractive index and any surface-bound adlayer(s)swill result in a phase change in the guided light. The effective penetration depth of the (16) Cross, G. H.; Reeves, A.; Brand, S.; Swann, M. J.; Peel, L. L.; Freeman, N. J.; Lu, J. R. J. Phys. D: Appl. Phys. 2004, 37, 74-80. (17) Cross, G. H.; Reeves, A.; Brand, S.; Popplewell, J. F.; Peel, L. L.; Swann, M. J.; Freeman, N. J. Biosens. Bioelectron. 2003, 19, 383-390.

evanescent field is 100 nm. The light in the lower waveguide experiences no such influence, i.e., experiences a constant refractive index environment and thereby provides an optical “reference”. Thus, when the light exits from each of the waveguides, they are no longer in phase. By simply allowing the output light to diverge from the two waveguides and combine with each other at some distance (in the “far-field”), a series of light and dark bands (interference fringes) is observed. The precise position of the light and dark bands depends upon the phase relationship of the light as it emerges from the two waveguides. As adlayers are added or removed from the sensor surface, or change in thickness, the relative phase difference between the light exiting the two waveguides changes, and hence, the position of the interference fringes moves. The phase changes are deduced from the distance of fringe movement. A second measurement is provided by introducing a second polarization of light, at right angles to the first into the stack (i.e., transverse magnetic (TM) compared to transverse electric (TE) polarization). This responds differently to adlayer adsorption/ desorption due to the differing penetration depths of the evanescent wave that is polarization dependent and thereby provides an independent second measurement. Using classical optical theory, it is possible to interpret the two measurements in terms of a thickness and refractive index for an adsorbed protein film modeled as a homogeneous slab adlayer. Using this technique, measurements to subatomic resolutions in protein layer thickness can be recorded in real time. It is important to recognize that this measurement embodies a quantitative analytical technique rather than a simple “sensor” response function, providing absolute measurements that can be related directly to the structure and function of materials immobilized in proximity to the measurement surface. We describe this analytical technique as dual polarization interferometry. EXPERIMENTAL DETAILS All chemicals and biochemicals were purchased from Sigma Aldrich (Poole, UK) unless otherwise stated. Sensor chips comprising a silicon oxynitride surface modified to introduce covalently bound amine groups (FB 100 Amine chips, Farfield Scientific Ltd.) were used for all experiments. The amine functionalization was achieved using silane chemistries adapted from the literature.18 Each chip has two areas that have exposed waveguide surface and that are allowed to contact individual or common liquid samples via a two-channel microfluidics cell. These are termed channel 1 and channel 3 with an additional waveguide area, channel 2, where the waveguide is not exposed and acts as an instrument reference channel, i.e., has a dielectric cover with constant refractive index. All experiments were performed with a temperature control to 0.002 °C and with a set point of 20 ( 0.01 °C using the temperature control built into the AnaLight Bio200 instrument. A simple fluidics system with Rheodyne HPLC injector valve and external pump (MillliGAT, Global FIA) enabled a controlled continuous fluid flow that could be directed through either, or both, experimental fluidic channels and also contained an injection-loop system to allow delivery of fixed volumes. An initial procedure to calibrate each sensor chip was performed as follows. A phosphate-buffered saline pH 7.4 solution

(PBS) was passed through both channels at a flow rate of 50 µL‚min-1. This was followed by a calibration solution (8:2 ethanol/ water mixture by weight) at the same flow rate for 2 min before returning to PBS. After 3.5 min, 50 mM, pH 7.6 tris(hydroxymethyl)aminomethane (Tris) buffer was injected for 3 min before returning again to PBS. This short procedure enabled the refractive index response of the chip to be calibrated. The values obtained were used by the analysis software of the AnaLight Bio200 instrument in all subsequent calculations. Transglutaminase Immobilization Procedure. Both channels were subjected to 3.5 min of a 50 µL‚min-1 flow of PBS after the calibration procedure and followed by a solution of the homobifunctional cross-linker bis(sulfosuccinimydyl) suberate (BS3) (4 mg‚mL-1) (Perbio Science UK Ltd., Cramlington, UK) for 4 min before the flow rate was reduced to 10 µL‚min-1 for a further 8 min before returning to PBS. The “working” channels defined as the channel to which protein was to be immobilizeds was then selected, and a solution of tTG (from guinea pig liver, Sigma Aldrich product code T5398) (1 mg/mL in PBS) was injected at a flow rate of 50 0 µL‚min-1 for 2 min and then 10 µL‚min-1 for a further 8 min before returning to PBS at a flow rate of 50 µL‚min-1 for a further 5 min. To deactivate (block) remaining immobilized succinimydyl groups, a 3 M Tris solution was injected, and after 1.5 min, the “control” channel, i.e., channel with BS3 immobilized but without exposure to tTG, was also switched into the solution flow for a further 2.5 min. A further 4-min Tris block was applied to both channels after 3 min. Careful consideration was given to the use of a negative control surface. While a protein that is not capable of binding calcium could have been employed, it was considered that the high salt concentrations required in this investigation were likely to cause changes in the solvation status leading, potentially, to changes in surface structure. The control surface as used provides a faithful replication of the surface structure underlying the tTG surface and as such should be expected to exhibit any experimental artifacts that might arise. Subsequent experiments using similar immobilization and control protocols using another calcium-binding protein, calmodulin, have demonstrated that calcium binding affinity constants as a consequence of structural changes, which were 3 orders of magnitude lower than those described in the current investigation, could be determined19 suggesting that this is an acceptable experimental approach. Calcium Chloride and Sodium Chloride Injections. Before the various salt injections proceeded, the flow was stopped and the buffer changed from PBS to Tris buffer (50 mM, pH 7.6) at a flow rate of 50 µL‚min-1. This was achieved as follows: Tris buffer was introduced to the channels at 50 µL‚min-1 and the flow rate increased to 200 µL‚min-1 over a 3-min time period and left for 28.5 min at this high flow rate to allow the working and control surfaces to stabilize. The flow rate was then reduced to 50 µL‚min-1 over a 2-min period. The sensor system was then deemed ready for salt injections. Stock solutions of calcium chloride (30 mM) and sodium chloride (60 mM) were made up in Tris buffer solution and diluted to provide calcium chloride solutions in the range of 0.15-30 mM and sodium chloride solutions in the range of 0.32-60 mM (equimolar with respect to

(18) Cass, T., Ligier, F. S., Eds. Immobilised Biomolecules in Analysis: A Practical Approach; Oxford University Press: Oxford, 1998.

(19) Swann, M. J., unpublished results.

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Table 1. Concentrations of Calcium and Sodium Chloride Used When Injecting Progressively Higher Salt Concentrations injection number

[CaCl2]/mM

injection number

[NaCl]/mM

Ca1 Ca2 Ca3 Ca4 Ca5 Ca6 Ca7 Ca8 Ca9

0.15 0.32 0.63 1.25 2.5 5.0 10.0 15.0 30.0

Na1 Na2 Na3 Na4 Na5 Na6 Na7 Na8 Na9

0.32 0.63 1.25 2.5 5.0 10.0 20.0 30.0 60.0

chloride ion concentration). The concentrations are given in Table 1. The injection sequence began with the lowest concentration of calcium chloride and then the lowest concentration of sodium chloride and progressed through to the highest concentrations. The procedure was then reversed, injecting the highest sodium chloride concentration and then the highest calcium chloride concentration through to the lowest salt concentrations. Each salt injection lasted for 3 min, and at least 4 min of elapsed Tris buffer washing was carried out between injections. RESULTS AND DISCUSSION The data analysis is presented in three stages to demonstrate the analytical value of the data obtained, raw signal data, resolved data, and resolved difference data (the difference between a tTG and a control surface). Thus, it will be shown that the measured dimensional changes in tTG when it was challenged with calcium ions were clearly evident in the raw data and demonstrate the assumptions that are required to undertake more complex quantitative data analysis necessary to obtain the thickness, density, and therefore mass measurements of the immobilized protein film. The rationale adopted with respect to the experimental design and execution is also discussed. Raw Signal Data: TM Phase Responses. While the experimental data were recorded in both TM and TE polarizations, for clarity, the course of the experiment is shown below in solely TM phase change data. As a single measurand, this provides information on the effective refractive index changes occurring at the surface of the sensor as the experiment proceeds. This is sometimes referred to as the “optical mass”. The initial tTG immobilization process together with the control channel preparation is shown in Figure 2. The layers obtained can be seen to be strongly adhered to the surface, as there are significant residual phase changes after returning to running buffer solutions, indicating the presence of substantial mass of material remaining on the surface. At this stage, it is not, however, possible to make any assumptions regarding the structures adopted by these materials at the interface. Having prepared the surfaces, a sequence of injections proceeded with a range of sodium and calcium chloride solutions (see Figure 3). It can be seen that the profile of the injection sequences for both the sodium and calcium ion injections are similar on both the working surface (with immobilized tTG) and the control 3026 Analytical Chemistry, Vol. 79, No. 8, April 15, 2007

surface (without tTG). When the phase change responses to the working surface and the control surface are plotted, it can be seen that they are very similar (see Figure 4). These phase changes are predominantly due to the changes in bulk refractive index of the solution above the chip, which dominate the observed response. From this data presentation, it would therefore be difficult using this optical mass parameter to determine the nature of the changes occurring within the immobilized tTG layer during the calcium injections or to differentiate them from the sodium injections. Comparing Raw Phase Responses in Both TM and TE Polarizations. When we compare the phase data from both polarizations after subtraction of the control channel, it immediately becomes clear that there were differences between the sodium and calcium injections. The differences in phase shifts between the tTG surface and the control surface are shown in Figure 5. While the response in terms of the TM phase change between the two surfaces is small, there is a clear and significant difference when the phase shifts in the TE polarization are considered. It is therefore clear that there were significant differences in the measured responses when both polarizations were considered. The nature of these differences were then analyzed in more detail. Resolved Data Set. The data from both the tTG and the control surface were then “resolved” and expressed in terms of the thickness and refractive index (RI). The density of the layers can be calculated from the RI if a refractive index and density for the pure protein are assumed. The standard values of 1.465 for RI and 0.71 g‚cm-3 have been used in the subsequent calculations.20 Having measured the thickness and calculated the density, the data can optionally be presented in terms of the mass of the layers residing on the sensor surface (the mass being the product of the thickness and density of the layers). The results are examined in detail below. Protein Layer Structure. The detail of the layer deposition process onto the amine (sensor) surface and the structures obtained is much clearer when the data are resolved in terms of dimensions and density. tTG was immobilized on the working surface (see Table 2) and the control surface was constructed in the same way but with just BS3 and 3 M Tris block (data not shown). The layer parameters are based on phase changes observed from the chip in PBS, after ethanol and buffer injections and before the start of immobilization. In Table 2, the sequential layers are treated as a homogeneous structure. Layer 1 was a layer of BS,3 an amine-amine linker with a 1.1-nm spacer, and the observed layer thickness was consistent with the expected layer characteristics. tTG has a prolate ellipsoidlike shape, which is ∼11 nm in diameter by 6.2 nm by 4.2 nm.10 The layer thickness obtained after layer 2 had been added (∼4.6 nm) was consistent with the protein being immobilized long axis parallel to the sensor chip surface. The layer is a composite of the protein and the linker, and therefore as the refractive index and density of the thinner linker layer was greater, the overall measured thickness change on immobilization of the protein layer (tTG - BS3 ) 3.6 nm) is somewhat of an underestimate. The (20) Swann, M. J.; Peel, L. L.; Carrington, S.; Freeman, N. J. Anal. Biochem. 2004, 329, 190-198

Figure 2. Phase changes (transverse magnetic) during the layer deposition process on both the working and control channels. Injection sequence in PBS pH 7.4 running buffer as follows: 80% ethanol calibration (1 min); 50 mM Tris buffer pH 7.6 (6.5 min); BS3 (12 min); tTG (channel 1 only 28.25 min); 3 M Tris block and repeat (43.25 and 50.2 min); running buffer change to 50 mM Tris (73 min).

Figure 3. Phase changes (transverse magnetic) during the sequence of injections of sodium and calcium chloride solutions. The injection sequence was as follows: (1) 0.32 mM NaCl (6060 s); (2) 0.15 mM CaCl2 (6720 s); (3) 0.63 mM NaCl (7140 s); (4) 0.32 mM CaCl2 (7800 s); (5) 1.25 mM NaCl (8340 s); (6) 0.63 mM CaCl2 (8880 s); (7) 2.5 mM NaCl (9420 s); (8) 1.25 mM CaCl2 (9900 s); (9) 5 mM NaCl (10 500 s); (10) 2.5 mM CaCl2 (10 980 s); (11) 10 mM NaCl (11 460 s); (12) 5 mM CaCl2 (12 000 s); (13) 20 mM NaCl (12 600 s); (14) 10 mM CaCl2 (13 080 s); (15) 30 mM NaCl (13 680 s); (16) 15 mM CaCl2 (14 160 s); (17) 60 mM NaCl (14 760 s); (18) 30 mM CaCl2 (15 240 s).

layers can also be modeled separately as independent layers. The dimensions obtained remained consistent with the known characteristics of the molecular species, with the protein layer now being measured at 4.1 nm. After the Tris block in Table 2, the overall density of the layer (RI) is seen to decrease and the layer thickness to reduce slightly. This is predominantly due to Tris acting as a surfactant and removing physisorbed protein, effectively leading to a reduction in the density of the protein layer as would be expected. The surface layer structures for both the working and the control surfaces in Tris buffer are shown in Table 3. The refractive

index of the bulk buffer solution was calculated by measuring the transition between PBS and Tris buffers prior to protein immobilization. The layers can be seen to undergo minor structural changes that are likely to be due to changes in the solvated status of the layers themselves. (See Table 3 in comparison with the last row in Table 2.) From the mass loadings achieved, it is also possible to calculate the area per molecule surface occupancy, which provides a confirmation of whether or not the tTG layer was likely to have been a monolayer structure (though this is also likely from the measured thickness). The calculated area per molecule in the Analytical Chemistry, Vol. 79, No. 8, April 15, 2007

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Figure 4. Phase change responses (transverse magnetic and transverse electric) observed on the working (Ch1) and control (Ch3) surfaces for calcium and sodium chloride injections.

Figure 5. (A) Differences in TM phase response between channel 1 (tTG surface) and channel 3 (control surface) (B) Differences in TE phase response between channel 1 (tTG surface) and channel 3 (control surface). Table 2. Layer Structures for BS3 Layer, tTG, and tTG Post Tris Block in PBS pH7.4 Running Buffer As Resolved by Fitting Both Polarization Phase Data Sets to a Slab Adlayer Modela layer 1 2 3

material

RI

thickness/nm

mass/ng‚mm-2

BS3 1.4912 ((0.0001) 0.958 ((0.007) 0.8169 ((0.0005) tTG 1.4350 ((0.0001) 4.580 ((0.007) 2.5025 ((0.0005) Tris block 1.4302 ((0.0001) 4.451 ((0.007) 2.3164 ((0.0005)

aShown in part in Figures 2 and 3. Errors correspond to standard deviation of the baseline noise obtained from the phase measurements.

experiment based on a protein molecular mass of 76.6 kDa was 76 nm2, which suggests that the protein layer was indeed a monolayer. Calcium and Sodium Injections. In determining the changes that occur in the tTG layer during the salt injections, the most direct approach, which essentially removes the contribution from the bulk refractive index, is to “resolve” the thickness and RI 3028 Analytical Chemistry, Vol. 79, No. 8, April 15, 2007

Table 3. Layer Structure for Entire BS3/tTG Layer in Tris Running Buffer Compared with Control Surface As Resolved by Fitting Polarization Phase Data to a Slab Adlayer Modela layer

material

RI

thickness/nm

mass/ng‚mm-2

1 2

Working Surface with Protein Tris buffer 1.3337 ((0.0001) n/ab n/a tTG (in Tris) 1.4184 ((0.0001) 5.261 ((0.007) 2.4105 ((0.0005)

1 2

Tris buffer BS3 + Tris

Control Surface without Protein 1.3337 ((0.0001) n/a n/a 1.4533 ((0.0001) 1.698 ((0.007) 1.0984 ((0.0005)

aTaken from Figures 2 and 3, b Errors correspond to standard deviation of the baseline noise obtained from the phase measurements. c n.a, not available.

values from the differences in the phase values from the working and control channels, and this approach is used below. Resolved Data after Control Channel Subtraction. The sodium and calcium ion binding data were analyzed in terms of

Table 4. Layer Parameters after Subtraction of the Control Channela layer 1

material tTG

RI

thickness/nm

mass coverage/ / ng‚mm-2

1.3885 ((0.0001)

4.395 ((0.007)

1.3040 ((0.0005)

a Taken from Figures 2 and 3. Errors correspond to standard deviation of the baseline noise obtained from the phase measurements.

differences occurring directly between the working and control surfaces. The phase value on the control channel was first normalized according to the magnitude of the ethanol injections on the two channels, in order to account for any small variation in the sensitivity between the two channels. In Table 4, the thickness, density, and mass values are shown for the tTG immobilization when subtracting the control channel phase values from those obtained on the working surface. The mass value is somewhat lower than the values obtained in Tables 2 and 3. This is due to the interaction of Tris on the control surface, which would be expected to be somewhat greater than on the working surface resulting in a slight underestimate of the tTG layer mass. These

errors are less than 20% and do not detract from the overall quality of the analysis. In Figure 6, the surface structure changes occurring when the tTG surface is challenged with sodium and calcium ions (equimolar with respect to chloride ions) is compared (after control channel phase subtraction). The main assumption is that the bulk change experienced by the control is the same as that observed on the working channel (including any surface charging phenomena). This in general means that there is a very small overestimate of the bulk refractive index contribution as the tTG layer acts to reduce these effects. These data show the direct changes in the mass, thickness, and index of the layers, which are actually experienced at the sensor surface. The effect of calcium challenge on the tTG layer can be seen to cause a nonlinear concentration-dependent decrease in the thickness of the layer and concomitant increase in the refractive index (density). The sodium injection show much smaller changes, which occur predominantly at higher concentrations. The mass changes are very small by comparison with the thickness and RI changes. The mass decreases with increasing concentration for both sodium and calcium ions, though the calcium line deviates from the sodium line at lower concentrations with a small mass

Figure 6. Changes in mass, thickness, and RI for calcium and sodium injections.

Figure 7. (Left) Changes in the thickness of the tTG layer as a function of calcium chloride injections ranging from 0.15 to 30 mM. (Right) Changes in the thickness of the tTG layer as a function of sodium chloride injections ranging from 0.32 to 60 mM.

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Figure 8. Thickness, refractive index, and mass loading difference plot (calcium - sodium) against chloride ion concentration.

Figure 9. Plot of layer refractive index, using data from Figure 8, against calcium ion concentration and fitting of data to a simple receptor binding model. Fitted parameters: KD ) 0.95 mM; ∆RImax ) 0.0102.

gain and remains above the sodium line at higher concentrations. The downward slope observed in the thickness of the tTG layer with increasing sodium concentration (or calcium at higher concentration) is likely to be a consequence of the small difference in the contribution of bulk refractive index and extent of surface charging occurring between the reference and working channels. This is also likely to be the cause of the decrease in the mass observed for both as there is probably a small increase in the surface ion structure of the chip with increasing salt concentration. This is more marked on the control channel, which causes a decrease in the net working channel response at higher concentrations. Figure 7 shows the real-time thickness changes for the endpoint data plotted in Figure 6. Using NaCl there is no obvious correlation between the salt concentration and the observed thickness of the tTG layer until the very highest (greater than 10 mM) concentrations. This effect is likely to be due to anomalous 3030 Analytical Chemistry, Vol. 79, No. 8, April 15, 2007

charging effects at these high concentrations. In the case of the calcium injections, there is a clear correlation between the concentration of the salt concentration and the observed tTG layer dimensions, the layer becoming thinner as the calcium concentration increases. The kinetics of the binding event and the dissociation are rapid, taking less than 60 s to reach equilibrium with similar dissociation rates. In Figure 8, the effects of sodium are subtracted from the calcium responses, as a means of accounting for the differences in surface charging between the channels. It again clearly shows that tTG surface contracts and densifies as a consequence of being subjected to calcium. The thickness decreases by ∼0.4 nm (depending upon the concentration) and the refractive index increases by 0.008 while the mass loading increases by 0.004 ng‚mm-2 using the same constants as are used for the protein density and refractive index. This gives a mass increase per tTG molecule of 183-236 Da, and if one assumes the binding of a

calcium ion is associated with two chloride counter ions and therefore a total mass of 111 Da, this converts to ∼1.7-2.1 calcium ions bound per tTG molecule. This should be treated with considerable care as this makes the assumption that the sodium injections are causing the same displacement of counterions from the surface as is the case with calcium and that the sodium is not perturbing the binding site status. It also assumes that there no other ions resident in the binding site, which would subsequently be displaced in the presence of calcium ions. Information has been provided on the structure and the integrity of the immobilized protein. The analytical information obtained provides confidence in the nature of changes occurring in the protein layer, its orientation, and the likely distortion that the molecule has undergone as a consequence of the binding event. It has been shown that it would not be possible to obtain this information from the TM data (the equivalent of “optical mass”) alone because, while significant structural changes are taking place, the overall mass change is very small. In the case of optical mass-based changes, structural changes cannot be measured directly, putative structural changes typically being reported as a consequence of deviations from expected responses.13 Correlation with Known tTG Parameters. In order to determine whether the differences observed between calcium and sodium were as an explicit consequence of calcium binding, the results were compared with known characteristics of the phenomenon. The affinity constant for tTG binding calcium has been measured by a number of workers, and values of between 0.2 and 3.0 mM have been reported.21 Thus, the affinity constant for the “interaction” was determined from the data of Figure 8. Affinity constants from the layer thickness and refractive index data were thus derived and compared. The plot for layer refractive index is shown in Figure 9. While Figure 8 assumed that sodium had the same bulk and surface charge effect as calcium (at the same chloride concentrations), as has already been noted, the refractive indices of the calcium and sodium salts while similar are not

identical, and small differences in the refractive index are evident at higher concentrations. These have been accounted for by subtracting a linear offset (labeled Ca-NaCorr in Figure 9). A curve, assuming a single binding site according to eq 1, was then fitted to the plot from which the affinity constant was derived.

(21) Mottahedeh, J.; Marsh, R. J. Biol. Chem. 1998, 273, 29888-29895.

AC051254B

nlayer ) nmax - 1/(1 + KD/[Ca2+])

(1)

The affinity constant was calculated to be 0.95 mM using this method. A slightly lower value of 0.74 mM (∆Thmax ) -0.55) was calculated when the thickness value (plot not shown) was used (the alternative approach for analysis without subtraction of the control channel, mentioned earlier, provided values of 1.02 mM from the RI and 1.16 mM from the thickness). These results suggest that the assumption that the binding sites are equivalent is reasonable and are in agreement with values reported in the literature. CONCLUSIONS In this paper, we have reported the observation and analysis of conformational structural changes occurring in an immobilized tTG layer using DPI. The structural changes occurring as a consequence of tTG binding calcium ions are clearly evident. A number of assumptions were made in order to obtain analytical data, which is found to be in agreement with known characteristics of the protein and binding events under investigation, typical tTG layers being of the order of 5 nm in thickness. It has been possible to determine the magnitude of the structural changes occurring in real time as a function of calcium ion concentration, the films typically contracting by ∼0.5 nm depending on the calcium concentration, and to estimate the possible stoichiometry of the binding event (1.7-2.1 calcium ions bound/ tTG molecule). Received for review July 14, 2005. Accepted January 11, 2007.

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