Fingerprints of Calcium-Binding Protein Conformational Dynamics

Jul 5, 2016 - Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, I-37134. Verona ...
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Fingerprints of calcium-binding protein conformational dynamics monitored by surface plasmon resonance Daniele Dell'Orco, and Karl-Wilhelm Koch ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00470 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

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Fingerprints of calcium-binding protein conformational dynamics monitored by surface plasmon resonance Daniele Dell’Orco1* and Karl-Wilhelm Koch2 1

Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, I-37134 Verona, Italy

2

Department of Neurosciences, Biochemistry Group, University of Oldenburg, D26111 Oldenburg, Germany * to whom correspondence should be addressed: strada le Grazie 8, I-37134 Verona, Italy. Telephone: +39-045-802-7637; e-mail: [email protected]

Abstract Surface plasmon resonance (SPR) spectroscopy is widely used to probe interactions involving biological macromolecules by detecting changes in the refractive index in a metal/dielectric interface following the dynamic formation of a molecular complex. In the last years SPR-based experimental approaches were developed to monitor conformational changes induced by the binding of small analytes to proteins coupled to the surface of commercially available sensor chips. A significant contribution to our understanding of the phenomenon came from the study of several Ca2+-sensor proteins operating in diverse cellular scenarios, in which the conformational switch is triggered by specific Ca2+ signals. Structural and physicochemical analyses demonstrated that the SPR signal not only depends on the change in protein size upon Ca2+-binding but likely originates from variations in the hydration shell structure. The resulting changes in the dielectric properties of water or of the protein-water interface eventually reflect different crowding conditions on the SPR sensor chip, which mimic the cellular environment. SPR could hence be used to monitor conformational transitions in proteins, especially when a significant variation in the hydrophobicity of the solvent-exposed protein surface occurs, thus leading to changes in the dielectric milieu of the whole sensor chip surface. We review recent work in which SPR has been successfully employed to provide a fingerprint of the conformational change dynamics in proteins under native and altered conditions, which include posttranslational modifications, co-presence of competing analytes and point mutations of single amino acids associated to genetic diseases.

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Introduction Surface plasmon resonance (SPR) is the optical phenomenon referring to resonant collective oscillations of valence electrons in a solid stimulated by light. When the frequency of the incident light matches the natural frequency of the oscillating surface electrons, resonance occurs.1 In the last decades, commercial and in-house built devices have been developed, which exploit this phenomenon at the interface between a thin metal surface, normally gold or silver, and an aqueous medium with different refractive index. When biological macromolecules are adsorbed on the surface, this will cause changes in the local refractive index, thus changing the resonance conditions of the surface plasmon waves and allowing the real-time detection of the adsorption process.2 The SPR-based technology has been widely used to study the interaction of proteins, oligonucleotides, carbohydrates, lipids and vesicles with other biological macromolecules or small ligands, and the kinetic parameters assessed to quantify the binding processes.3, 4 Despite its broad applications in biochemical and biophysical research, SPR has been only occasionally used to monitor conformational changes in proteins immobilized on the surface of SPR-devices. One reason for SPR not being widely used in monitoring protein conformational changes is that several other experimental techniques are available and have been demonstrated being highly effective. X-ray crystallography provides atomisticresolution pictures of macromolecules in specific conformational states, although it is limited to static conformations of crystallized molecules. Nuclear Magnetic Resonance (NMR) is highly suitable for studying the dynamics of proteins in solution and their conformational properties, but physical limitations are the size of the protein and the significant amount needed. Other lower-resolution techniques, which are widely used to study conformational properties of proteins in solution include fluorescence spectroscopy, either intrinsic or based on dyes, circular dichroism and dynamic light scattering. Often each of the aforementioned techniques is well suited to answer specific questions on protein structure, and thus they are used in combination in biochemical and biophysical studies. A unique advantage of SPR-based technology is that it requires extremely low amount of protein to be immobilized, down to a few nanograms. Other advantages, yet shared by other techniques, are: i) the completely label-free approach and consequent minimal perturbation of the system under investigation; ii) the possibility, for many of the commercially available systems, to work without direct illumination of the sample, with clear benefit for molecules subject to bleaching; and iii) the real-time recording, that allows to monitor the kinetics of the processes under investigation. It is apparent that the possibility to monitor protein conformational dynamics at real-time in label-free conditions, using extremely low amount of proteins is appealing, and attempts to employ SPR for this specific task were made already in the nineties (see the references in the next paragraph). However, some intrinsic difficulties and apparent misinterpretations of the results emerged rather soon after the first applications, and somehow slowed down the progress in the field until the very last years. In this review, we will summarize recent results in the development and applications of SPR-based strategies to monitor the dynamics of protein conformational changes induced by the binding of small analytes, a topic of wide interest in chemical biology applications.

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Conformational changes detected by commercially available and in-house built biosensors Among the first applications of SPR to detect protein conformational changes, Salamon et al.5 reconstituted the integral membrane protein rhodopsin into a phosphatidylcholine bilayer that was directly immobilized on a silver film, and were able to measure its light-induced conformational change. The approach has been extended by Tollin and co-workers, who developed a plasmon waveguide resonance (PWR)-based device specialized in monitoring conformational changes occurring in integral membrane proteins incorporated into supported lipid bilayers, by taking advantage of both p- and s- polarized light, i.e. monitoring conformational changes occurring both perpendicular and parallel to the resonator surface, respectively.6 By a different approach, Boussaad et al.7 used a self-assembled monolayer of 3mercaptopropionate to immobilize a monolayer of cytochrome c and monitor conformational changes following variations of the redox state. The use of commercially available sensor chips to study the conformational changes induced on the immobilized proteins appeared relatively recently in the literature. In these studies, the proteins of choice were immobilized on a carboxymethylated dextran (CMD) surface that is coated on a thin gold film available as commercial sensor chips (CM5, Biacore). By this procedure the conformational changes of phenylalanine hydroxylase,8 maltose-binding protein and transglutaminase,9 dihydrofolate reductase10 and α-glucosidase11 were investigated. A different aspect of the phenomenon was reported in a study highlighting a relationship between the hydrophobicity of the immobilized protein and the amplitude of the detectable signal.12, 13 Earlier studies employing commercially available sensor chips have been critically reviewed by Winzor, who highlighted the presence of apparent artefacts in some cases.14 In particular, it was noticed that in some studies the effect attributed to protein isomerisation followed by pH changes was instead due to electrostatic interactions between the immobilized protein and the CMD matrix, which were therefore nonspecific.15 In-house built devices using the SPR principle have also been recently employed to investigate protein conformational changes. For example, the antibody chip technology developed by Chung and co-workers has been used to monitor conformational alterations triggered in the Bax protein by an apoptosis inducer,16 while the approach developed by Chen to monitor RNase and lysozyme chemical unfolding/refolding provided thermodynamic insight into the energetics of immobilization/conformational change.17 More recent applications concerned the study of the Alzheimer disease-related β-amyloid peptide aggregation as regulated by copper and zinc ions, which have been shown by Yao et al. to induce conformational changes in the peptide that can be detected by SPR.18

Monitoring Ca2+-induced conformational changes in Ca2+ sensor proteins by SPR An ideal system to investigate in more detail the potential of SPR in detecting protein-specific signals related to conformational dynamics is represented by Ca2+sensor proteins. This important class of protein sensors switch conformation in response to variations in the Ca2+ concentration and selectively bind specific targets

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thus regulating a number of biological processes. Conformational dynamics of Ca2+sensor proteins have been investigated by a number of biophysical techniques,19 but when it comes to SPR mainly two different experimental setups were used. One setup consists of nano-sized gold or silver particles coated with the protein of interest,20, 21 while in the other setup proteins were immobilized on commercial sensor chip surfaces and SPR signals were recorded by Biacore devices.22-27 In recent studies, the van Duyne group developed nano-sized sensors that, based on the local SPR (LSPR) phenomenon were able to detect conformational changes in a surface bound construct of the 17 kDa calcium binding protein calmodulin (CaM).20, 21 CaM was sandwiched between two higher molecular weight (22 kDa) cutinase domains coupled to a phosphonate functionalized Ag-nanoprism. Upon Ca2+-binding the protein packing density close to the sensor surface changes due to reversible conformational changes in CaM. The shifts in the maximum wavelength of LSPR in response to changes in Ca2+ concentration were found to be 2-3 nm, in response to alternating cycles of 2 mM CaCl2/EGTA. The concentrations of Ca2+ and EGTA pulses used in the experiments significantly exceeded the apparent affinity of CaM for Ca2+ (KD 1 mM Ca2+ (Figure 2A). Since these SPR responses cannot origin from a Ca2+-triggered conformational change in RecE121Q, data interpretation in previous reports might be wrong. It remains challenging to distinguish a real binding process involving the immobilized protein from nonspecific, matrix-related effects due to changes in pH and/or ionic strength. In a recent work to probe whether the experimental setup could be used in general to study conformational changes induce by Ca2+ in diverse protein systems, Dell’Orco et al.24 immobilized on the same surface of a CM5 sensor chip four different calcium sensor proteins involved in the vertebrate phototransduction cascade in rod cells, in which the concentration of Ca2+ drops from 0.6 μM to 0.1 μM upon illumination of the photoreceptor cell as a consequence of complex biochemical events.37, 38 The four proteins were: Rec, two guanylate cyclase activating proteins (GCAP1 and GCAP2) and CaM. It should be noticed that all four proteins are set to physiologically work in the same cell regulating biochemical processes while Ca2+ changes dynamically upon illumination, and their Ca2+-sensitivity is limited to a narrow range of concentration. While Rec undergoes the already mentioned Ca2+-myristoyl switch, GCAP1 and 2 respond differently to Ca2+ binding with a smaller conformational arrangement around the myristoyl group.39-44 CaM on the other hand is not myristoylated, but exhibits distinct conformational shapes in its Ca2+-bound and -free

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forms as discussed above. The system therefore constituted an ideal benchmark for probing the SPR-based strategy in detecting conformational changes triggered by fine differences in Ca2+ sensitivity, in proteins that are expected to respond differently to Ca2+-signals. In order to distinguish the effect of Ca2+ on triggering a conformational change from its direct binding to the Ca2+-sensor proteins, Ca2+binding was also measured by an established titration method based on the competition for Ca2+ between the protein and a chromophoric chelator.45-47 Binding measurements were performed both in the absence and in the presence of 1 mM Mg2+, corresponding to the physiological free levels found in rod cells. The analysis of the equilibrium data obtained by SPR (K1/2SPR) for cases in which saturation was clearly observed and the Ca2+-binding affinity directly measured by the chelator assay (KDapp) allowed the estimation of the conformational change contribution (Kcc) in the concerted binding-conformational transition process (Ktot), from the relationship: Ktot = 1/ K1/2SPR = Kcc / KDapp

(1)

This comprehensive analysis allowed to distinguish cases in which Mg2+ exerted a pure electrostatic screening on Ca2+-binding from others, in which the whole dynamics of conformational changes are significantly altered, probably because of direct competition with Ca2+.24 As an example, the data obtained for CaM are reported in Figure 1B. Thus, this SPR approach is suitable to distinguish the effects Ca2+ exerts on protein conformation from the pure binding steps and is in full agreement with the known structural features of the protein.17 To our knowledge it represents the first attempt to dissect these two features (conformational change versus binding) by employing SPR. Testing the effects of competing analytes, ionic strength and point mutations on protein conformational changes detected by SPR An important addition to previous work was the investigation of the influence of specific metal cations on the detected conformational dynamics. Ca2+-titrations on immobilized Rec were followed in the presence of other salts at concentrations yielding the same ionic strength as 1 mM Mg2+, namely 2 mM KCl and 1 mM ZnCl2 were added to the running buffer and to the Ca2+ stocks to be injected (Figure 3). The addition of 2 mM K+ reduced the amplitude of each individual response by approximately 8%, hence suggesting a pure electrostatic screening effect as the only consequence. Significantly higher reduction of the maximal amplitudes ( 50%) was observed in the presence of 1 mM Mg2+, but neither of the added cations significantly shifted the measured K1/2SPR values. Interestingly, Ca2+-titrations performed in the presence of 1 mM Zn2+ did not cause any detectable change in the SPR signal , which is consistent with the known affinity of Rec for Zn2+ with an apparent KD of 30 μM for the apo-state.48 Saturation of Rec with Zn2+ may favour a conformation that is incompatible with the operation of the Ca2+-switch clearly visible with all other cations. Figure 3

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Figure 3. Sensitivity of the SPR-based strategy to ionic strength and protein posttranslational modifications. A) Effect of increased ionic strength on the SPR detection of conformational changes in Rec. The Ca2+-titration was repeated with the same immobilized sample in normal running buffer (●) (n = 2); in the presence of additional 2 mM K+ (□) (n = 3); in the presence of 1 mM Mg2+ () (n = 4); in the presence of 1 mM Zn2+ (◊) (n = 3). Error bars stand for the SD. B) Detection of conformational changes in nonmyristoylated recoverin (approximately 6 ng/flow cell 2+ immobilised) upon Ca -titration at T = 25 °C. Adapted with permission from Daniele Dell’Orco, 2+ Stefan Sulmann, Sara Linse, Karl-Wilhelm Koch, Dynamics of conformational Ca -switches in signalling networks detected by a planar plasmonic device, AnalChem 2012, 84, 2982-2989. Copyright 2011, American Chemical Society

Results summarized in Figure 3 and 4 also show that carefully performed experiments using commercially available sensor chips are sensitive to other factors which are relevant in chemical biology studies, such as posttranslational modifications or presence of point mutations. As an example, a dramatically changed SPR pattern was observed when the same Ca2+ titrations were performed with non-myristoylated Rec (compare Figure 3B with Figure 1A). Single point mutations of the Ca2+ sensor GCAP1, which cause cone dystrophy in patients with impaired vision46, 49 showed a variant specific pattern of SPR responses (Figure 4).26 The differential Ca2+-response pattern of GCAP1 mutants correlated to results

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obtained with dynamic light scattering and size-exclusion chromatography leading to the conclusion that changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric properties of water or of the protein-water interface.26 Figure 4

Figure 4. Examples of sensorgrams obtained by injecting increasing [Ca2+] in the 0.3–46 μM range on the surface of a sensor chip in which equal amounts of GCAP1 were immobilized. Each panel refers to a specific GCAP1 mutant involved in cone/rod dystrophy. Adapted with permission from Stefan Sulmann, Daniele Dell’Orco, Valerio Marino, Petra Behnen, Karl-Wilhelm Koch, Conformational Changes in Calcium-Sensor proteins under Molecular Crowding Conditions, Chemistry 2014;20(22):6756-62. Copyright 2014, Wiley

Exploiting the sensing volume of commercially available sensor chips confers high sensitivity and mimics the conditions found within cells It has been argued that the rather long (≈200 nm) decay length of the evanescent electromagnetic field (EM) in commercially available SPR sensor chips, such as the CM5 by Biacore, dramatically reduces the sensitivity to small refractive index changes occurring close to the sensor surface.21 This feature would lead to failure in detecting conformational changes in protein monolayers by such planar devices, a limit that is bypassed by nanodevices exploiting LSPR, which has a much shorter (510 nm) decay length.21 In order to probe directly the sensitivity of commercially available planar devices in this respect, Dell’Orco et al.24 performed experiments as summarized in Figure 5. The same rather low level of Rec (about 1 ng/flow cell) was covalently immobilized on the surface of two different commercially available sensor chips using the same amine coupling chemistry. In the first experiment however the already described CMD coated sensor chip was used, thus exploiting a ca. 100 nm long dextran matrix extending in the 0.02 µL volume of the flow cell. In another experiment, a dextran-free chip was used instead and the same amount of protein was immobilised on a flat carboxylated gold surface. When subjected to identical injections of 0.3–46 μM Ca2+, the two sensor chips showed a significantly different

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behaviour. When Rec was immobilized within the whole volume of the dextran matrix a reproducible and typical signal was detected in response to Ca2+ pulses, while in the case of adsorption on a two-dimensional surface, Ca2+ injections led to a monotonic sequence of signals overlaying a rapid drift of the baseline most likely due to fast protein loss and degradation (Figure 5A). Therefore, rather than a limit, the possibility to exploit the extension of the dextran matrix in its entirety constitutes an advantage for the tested sensor chips as it provides significantly increased sensitivity. The sensitivity can be in principle increased by increasing the amount of protein immobilised. In another set of experiments performed with Rec, it was in fact shown that reducing the amount of immobilized protein merely resulted in an overall decrease of the observed SPR signal (Figure 5B), while the shape of the curves remained the same. The striking linear correlation found between the maximum amplitude observed in Ca2+ titrations of Rec and the level of protein immobilised (Figure 5C, R2 = 0.99) suggests that increasing such amount in the range 1–10 ng/flow cell leads to better signal-to-noise ratio without perturbing the observed dynamics.24 Figure 5

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Figure 5. Dependence of the sensitivity of the SPR-based detection of protein conformational changes on the sensing volume and the amount of immobilised protein. A) Schematic representation of a portion of the surface of a CMD commercially available sensor chip, in which an approximately 100 nm thick dextran matrix extends from the thin gold film surface (left). A low amount (≈1 ng) of protein (red spheres) has been immobilized within the matrix exploiting the 2+ 0.02 µL volume of the flow cell, giving rise to the sensorgram shown above in response to Ca injections in the 0.4-46.2 μM range. Immobilization of the same protein on the flat carboxylated 2+ gold surface (right) lacking the dextran matrix leads to no specific signal in response to Ca injections (see the sensorgram above). The intensity of the electromagnetic evanescent field, represented as a red background cloud, decays almost completely 200 nm away from the gold surface, hence conferring great sensitivity to variations of refractive index in the whole volumetric extension of the flow cell. B) Different amounts of myristoylated Rec ((●) 8453 RU, (□) 4885 RU, (▼) 1740 RU; () 860 RU) were immobilised on a 100 nm CMD-coated sensor chip and titrated with increasing [Ca2+] in the 0.3–46 μM range. Titrations were repeated three times (n = 3) and data were fitted according to a Hill-sigmoidal. C) Averaged maximal response upon titration 2 plotted against the amount of immobilized myristoylated Rec. The correlation coefficient is R = 0.99. Adapted with permission from Daniele Dell’Orco, Stefan Sulmann, Sara Linse, Karl-Wilhelm 2+ Koch, Dynamics of conformational Ca -switches in signalling networks detected by a planar plasmonic device, AnalChem 2012, 84, 2982-2989. Copyright 2011, American Chemical Society

Relative advantages of SPR in monitoring protein conformational changes and possible future targets In spite of the low amount of protein immobilized, the extremely small volume (2050 nL) of the flow cells in commercially available sensor chips permits to monitor the conformational change dynamics under cellular-like conditions. Proteins can in fact be immobilized at high density in the polymeric matrix coating the sensor surface thereby reaching the micromolar cellular concentration. The use of sugar-based matrices such as dextran, itself used as crowding agent in experimental approaches,50, 51 allows one to simulate the macromolecular crowding conditions in cells and its potential effects on the processes under investigation. Reaching the same conditions with other spectroscopic techniques would require significantly higher amounts of protein. The possibility to exploit the flow of the running buffer confers another unique advantage to the commercially available plasmonic devices. The kinetics of conformational changes can indeed be monitored by simply injecting the analyte that triggers the transition, and the process can be reversed by simply flowing the running buffer, without interventions of other chemicals that may perturb the established interactions and thus the signal. The flow can also be employed to present the same analyte to different proteins immobilized on different flow cells, in order to monitor in parallel the induced changes. Finally, while the focus in the last years has been on Ca2+-binding proteins, we envision a fruitful applicaton of the same SPR approaches reviewed here to monitor conformational changes in metalloproteins. The advantage of using plasmonics technology for studying metal ion-protein interactions has been recently reviewed,52 and it is very likely that benefit will arise especially from the application of localized SPR approaches. Free-solution interactions studies performed by label-free approaches such as the SPR methodologies reviewed here and the recently developed backscattering interferometry assays53, 54 can be eventually used to yield a fingerprint profile of the protein under study that reflects different hydrodynamic properties under changing conditions. Such methods have been shown to be extremely sensitive to even fine

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alterations induced by point mutations, and are therefore expected to be of great relevance for general chemical biology purposes as well as biomedical oriented research.

Acknowledgments This work was supported by grants from the Italian Ministry for Education and Research via departmental funding (FUR2014 to DDO) and by grants from the Deutsche Forschungsgemeinschaft (KO948/10-1 and KO948/10-2 to KWK). References 1. Kooyman, R. P. H., Physics of Surface Plasmon Resonance. RSC Publishing: 2008. 2. de Mol, N. J.; Fischer, M. J., Surface plasmon resonance: a general introduction. Methods Mol Biol 2010, 627, 1-14. 3. Koch, K. W., Surface Plasmon Resonance. In Encyclopedic Reference of Genomics and Proteomics in Moelcular medicine, Springer: 2006; pp 1832-1835. 4. Vachali, P. P.; Li, B.; Bartschi, A.; Bernstein, P. S., Surface plasmon resonance (SPR)-based biosensor technology for the quantitative characterization of proteincarotenoid interactions. Arch Biochem Biophys 2015, 572, 66-72. 5. Salamon, Z.; Wang, Y.; Brown, M. F.; Macleod, H. A.; Tollin, G., Conformational changes in rhodopsin probed by surface plasmon resonance spectroscopy. Biochemistry 1994, 33, 13706-13711. 6. Tollin, G.; Salamon, Z.; Hruby, V. J., Techniques: plasmon-waveguide resonance (PWR) spectroscopy as a tool to study ligand-GPCR interactions. Trends Pharmacol Sci 2003, 24, 655-659. 7. Boussaad, S.; Pean, J.; Tao, N. J., High-resolution multiwavelength surface plasmon resonance spectroscopy for probing conformational and electronic changes in redox proteins. Anal Chem 2000, 72, 222-226. 8. Flatmark, T.; Stokka, A. J.; Berge, S. V., Use of surface plasmon resonance for real-time measurements of the global conformational transition in human phenylalanine hydroxylase in response to substrate binding and catalytic activation. Anal Biochem 2001, 294, 95-101. 9. Gestwicki, J. E.; Hsieh, H. V.; Pitner, J. B., Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance. Anal Chem 2001, 73, 5732-5237. 10. Sota, H.; Hasegawa, Y.; Iwakura, M., Detection of conformational changes in an immobilized protein using surface plasmon resonance. Anal Chem 1998, 70, 20192024. 11. Mannen, T.; Yamaguchi, S.; Honda, J.; Sugimoto, S.; Kitayama, A.; Nagamune, T., Observation of charge state and conformational change in immobilized protein using surface plasmon resonance sensor. Anal Biochem 2001, 293, 185-193. 12. Yamaguchi, S.; Mannen, T.; Nagamune, T., Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor. Biotechnol Lett 2004, 26, 1081-1086.

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13. Yamaguchi, S.; Mannen, T.; Zako, T.; Kamiya, N.; Nagamune, T., Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins. Biotechnol Prog 2003, 19, 1348-1354. 14. Winzor, D. J., Surface plasmon resonance as a probe of protein isomerization. Anal Biochem 2003, 318, 1-12. 15. Paynter, S.; Russell, D. A., Surface plasmon resonance measurement of pHinduced responses of immobilized biomolecules: conformational change or electrostatic interaction effects? Anal Biochem 2002, 309, 85-95. 16. Kim, M.; Jung, S. O.; Park, K.; Jeong, E. J.; Joung, H. A.; Kim, T. H.; Seol, D. W.; Chung, B. H., Detection of Bax protein conformational change using a surface plasmon resonance imaging-based antibody chip. Biochem Biophys Res Commun 2005, 338, 1834-1838. 17. Chen, L. Y., Monitoring conformational changes of immobilized RNase A and lysozyme in reductive unfolding by surface plasmon resonance. Anal Chim Acta 2009, 631, 96-101. 18. Yao, F.; Zhang, R.; Tian, H.; Li, X., Studies on the interactions of copper and zinc ions with beta-amyloid peptides by a surface plasmon resonance biosensor. Int J Mol Sci 2012, 13, 11832-11843. 19. Koch, K. W., Biophysical investigation of retinal calcium sensor function. Biochim Biophys Acta 2012, 1820, 1228-1233. 20. Hall, W. P.; Anker, J. N.; Lin, Y.; Modica, J.; Mrksich, M.; Van Duyne, R. P., A calcium-modulated plasmonic switch. J Am Chem Soc 2008, 130, 5836-5837. 21. Hall, W. P.; Modica, J.; Anker, J.; Lin, Y.; Mrksich, M.; Van Duyne, R. P., A conformation- and ion-sensitive plasmonic biosensor. Nano Lett 2011, 11, 10981105. 22. Christopeit, T.; Gossas, T.; Danielson, U. H., Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor. Anal Biochem 2009, 391, 39-44. 23. Dell'Orco, D.; Muller, M.; Koch, K. W., Quantitative detection of conformational transitions in a calcium sensor protein by surface plasmon resonance. Chem Commun (Camb) 2010, 46, 7316-7318. 24. Dell'Orco, D.; Sulmann, S.; Linse, S.; Koch, K. W., Dynamics of conformational Ca2+-switches in signaling networks detected by a planar plasmonic device. Anal Chem 2012, 84, 2982-2989. 25. Majava, V.; Loytynoja, N.; Chen, W. Q.; Lubec, G.; Kursula, P., Crystal and solution structure, stability and post-translational modifications of collapsin response mediator protein 2. FEBS J 2008, 275, 4583-4596. 26. Sulmann, S.; Dell'Orco, D.; Marino, V.; Behnen, P.; Koch, K. W., Conformational changes in calcium-sensor proteins under molecular crowding conditions. Chemistry 2014, 20, 6756-6762. 27. Sulmann, S.; Vocke, F.; Scholten, A.; Koch, K. W., Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+binding. Sci Rep 2015, 5, 11228. 28. Weinstein, H.; Mehler, E. L., Ca(2+)-binding and structural dynamics in the functions of calmodulin. Annu Rev Physiol 1994, 56, 213-236.

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