DOI: 10.1002/cphc.200900333

A --Based Single- Metal-Binding Assay

Yi Cao, Kai Shih Er, Rakesh Parhar, and Hongbin Li*[a]

Metal ions are crucial to the function of many machi- the mechanical stability of the given protein before and after neries inside cells.[1–3] They participate in catalytic cycles of en- the binding of its interacting protein partner. By extending this zymes,[4] mediate the protein–ligand interactions,[5,6] and main- methodology to metal-ion binding to , we report here tain the structure of proteins.[7] Determining the binding a force-spectroscopy-based single-molecule metal-ion-binding strength of metal ions to proteins, which is usually represented assay that is capable of determining the binding affinity of by the dissociation constant Kd, is thus of fundamental impor- metal ions to proteins at the single-molecule level. This novel tance for understanding the biological role of metals. Several assay does not require any assumption and can directly quanti- metal-binding assays—ranging from equilibrium dialysis, UV/ fy the partitioning of proteins between the apo and metal-ion- Vis spectroscopy, and isothermal titration calorimetry to capilla- bound states. As a proof-of-principle, we use the binding of ry electrophoresis—have been developed at the ensemble Ni2+ to an engineered metal-binding protein as a model level to determine Kd. These methods have been the dominant system to demonstrate that this new method can measure methods of choice in the studies of the binding of metal ions weak metal binding to proteins with a dissociation constant of to proteins. as high as 100mm. We anticipate that this method will comple-

The dissociation constant Kd can be calculated using the fol- ment the existing ensemble metal-binding assays and may lowing simple relationship:[8] Y ¼ ½M , where Y defines the find unique applications in a range of metal-binding systems, ½MþKd fraction of proteins bound to metal ions and [M] is the free including proteins, RNA and DNA, where traditional assays may metal-ion concentration at equilibrium. Direct measurement of become difficult to perform.

Y offers the most straightforward way to determine Kd. Howev- Herein, we use an engineered metal-binding protein, er, except for equilibrium dialysis, most metal-binding assays namely, G6-53, to demonstrate the proof-of-principle of the cannot directly measure Y at the ensemble level.[9] These bind- single-molecule force-spectroscopy-based metal-ion-binding ing assays rely on the relationships between Y and the change assay. The engineered metal-binding protein G6-53 is a bi-histi- of a particular thermodynamic parameter, such as the heat re- dine mutant of a small protein, GB1, in which residues 6 and leased or the change in thermodynamic stability upon binding 53 were mutated to histidines to form an engineered metal [14] of metal ions, to determine Kd. In some cases, certain assump- binding site. Our previous equilibrium denaturation experi- tions have to be taken to derive the relationship between Y ments showed that bi-histidine mutant G6-53 binds a variety 2+ 2+ 2+ 2+ and Kd. Although the thermodynamic properties used to deter- of divalent metal ions, including Ni ,Cu ,Zn , and Co . mine Kd in most metal-binding assays are distinct for the apo Single-molecule force spectroscopy experiments revealed that and metal-bound forms of the proteins, they can only be the binding of metal ions, such as Ni2+, can significantly en- measured as a convolution of the contributions from both the hance the mechanical stability of G6-53.[14] Figure 1 shows typi- apo and the metal-bound forms. The presence of any hetero- cal force-extension relationships of polyprotein (G6-53)8, which geneity in the binding complex will be averaged out in such is composed of eight identical tandem repeats of protein ensemble measurements. In contrast, single-molecule techni- G6-53, measured by using single-molecule atomic force micro- ques are uniquely well-suitable for directly identifying the apo scopy (AFM).[15,16] As shown before, stretching and unfolding of and metal-bound forms of proteins, thus enabling the direct polyprotein (G6-53)8 result in characteristic saw-tooth-like determination of Y and Kd. However, such efforts proved chal- force-extension curves, where individual saw-tooth peaks cor- lenging for studying the binding of metal ions to proteins, respond to the mechanical unfolding of G6-53 domains, and partly due to the fact that most binding events of metal ions the force at which G6-53 unfolds measures its mechanical sta- to proteins do not result in large conformational changes re- bility (Figure 1). Unfolding of G6-53 domains in the absence of quired by single-molecule techniques, such as single-molecule Ni2+ (Figure 1A) and in the presence of saturating concentra- Fçrster resonance transfer (FRET).[10–12] tions of Ni2+ (Figure 1B) results in unfolding events with simi-

Recently, we demonstrated the feasibility to use single-mole- lar contour length increment (DLc 18 nm) but different un- cule force spectroscopy as a functional assay to study protein– folding , which suggests that apo-G6-53 and Ni2+ -bound protein interactions.[13] This assay is based on the difference in G6-53 exhibit distinct mechanical stability: apo-G6-53 unfolds at around 120 pN (Figure 1A) whereas Ni2+-bound G6-53 un- [a] Y. Cao, K. S. Er, R. Parhar, Prof. H. Li folds at approximately 250 pN (Figure 1B). Thus, the distinct Department of Chemistry, The University of British Columbia mechanical stability between apo-G6-53 and Ni2+-bound 2036 Main Mall, Vancouver, BC, V6T 1Z1 (Canada) G6-53 offers a sensitive force probe to directly distinguish the + Fax: ( 1)604-822-2847 2+ E-mail: [email protected] apo and Ni -bound states of G6-53 in a mixture of both spe- 2+ Supporting information for this article is available on the WWW under cies when a non-saturating concentration of Ni is present in http://dx.doi.org/10.1002/cphc.200900333 the solution. Indeed, stretching polyprotein (G6-53)8 in the

1450 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2009, 10, 1450 – 1454 2 + Figure 1. The mechanical stability of G6-53 is a functional reporter for the binding of Ni to G6-53: A) Stretching polyprotein (G6-53)8 results in typical saw- tooth-like force-extension curves that are characterized by unfolding forces of about 120 pN (black line) and contour length increments DLc of approximately 18 nm. B) Ni2+-bound G6-53 domains exhibit a high mechanical stability. In the presence of a saturating concentration of N2 + (4mm), the unfolding of G6-53 m 2 + domains occurs at about 250 pN with a similar DLc value of about 18 nm. C) Unfolding of G6-53 domains in the presence of 0.2 m Ni occurs at two dis- tinct levels of forces (indicated by the dashed lines): one is at 120 pN and the other one at 250 pN. The DLc remains unchanged. The dashed lines are fits [27] to the experimental data obtained by using the worm-like chain model of elasticity. Insets: Schematic illustration of the stretching of (G6-53)8 poly- protein between an AFM tip and a glass substrate in the absence or presence of Ni2 +. presence of 200mm of Ni2+ results in saw-tooth-like force-ex- indicates that the statistics is not biased. Furthermore, we also tension curves with unfolding force peaks occurring at two dis- found that the success rate of picking up a polyprotein re- tinct levels of forces: one level is at about 120 pN and the mained the same ( 5%) for AFM experiments on the apo other one at about 250 pN. It is evident that the unfolding (0mm Ni2+) and bound forms (4mm Ni2+) of polyproteins. This force peaks at approximately 120 pN correspond to the me- suggests that there is no preference for one form (apo or chanical unfolding of the apo form of G6-53 domains, while bound) to attach to the tip. the unfolding force peaks at about 250 pN correspond to the By varying the concentration of Ni2+ , we can directly ob- mechanical unfolding of the Ni2+ -bound form of G6-53 do- serve a change in the partition of G6-53 between the apo and mains. Therefore, we can readily determine the Ni2+ -bound Ni2+ -bound forms. As shown in Figure 2, in the presence of a fraction of non-saturating concentration of Ni2+, the unfolding force his- G6-53 without using any binding model or prior knowledge. tograms of G6-53 show bimodal distributions, which can be For example, out of the seven G6-53 domains in Figure 1C, well-described by two Gaussian distributions (solid lines). Such three G6-53 domains are in their apo form and the other four bimodal distributions are in sharp contrast to the unimodal are in Ni2+ -bound form. The partitioning of G6-53 between the distributions of unfolding forces for apo-G6-53 and G6-53 in two forms is also evident from the unfolding force histogram the presence of a saturating concentration of Ni2+ . The popu- of G6-53 in the presence of 200mm of Ni2+, which shows a bi- lation at about 120 pN corresponds to the unfolding of the modal distribution with one peak at about 120 pN and the apo form while that at about 250 pN corresponds to the un- other one at about 250 pN (Figure 2). The number of events folding of the Ni2+-bound form. As expected, upon increasing falling under each peak directly measures the partitioning of the concentration of Ni2+, the unfolding events occurring at G6-53 between the apo and Ni2+ -bound forms. about 250 pN increase monotonically while those occurring at It is important to note that the histogram is built from indi- approximately 120 pN decrease accordingly. This indicates that vidual that have different number of G6-53 domains. the apo G6-53 domains are converted to the Ni2+-bound form This difference is due to the fact that the polyprotein was of G6-53 upon increasing the concentration of Ni2+ . The posi- picked up randomly along its contour in the AFM experiments. tions of the two unfolding force peaks in the histograms One concern is that the statistics might be biased if one form remain unchanged at different Ni2+ concentrations. (apo or bound) can be picked up preferentially by the AFM tip. The bimodal distributions of unfolding force histograms To resolve this issue, we carried out binding measurements at were built from individual molecules and no averaging was in- 0.2mm Ni2+on the same molecule, in which the number of volved. In the full range of Ni2+ concentrations we studied, no G6-53 domains remained the same. By repeatedly stretching additional peaks appeared in the unfolding force histograms, and relaxing the same molecule for hundreds of cycles, we de- thus suggesting that only two forms of G6-53 (the apo and termined the number of apo and bound forms of G6-53 in the Ni2+ -bound forms) exist in solution and that no binding inter- same polyprotein molecule, respectively. We found that the mediate states are present. Therefore, fitting the bimodal dis- measured partition between the apo and bound forms is indis- tributions to two Gaussian distributions allowed us to directly tinguishable from that measured from different molecules (see “count” the number of G6-53 domains in the apo form, which Figure S1 of the Supporting Information). This result strongly unfold at lower forces, and in the Ni2+ -bound form, which

ChemPhysChem 2009, 10, 1450 – 1454 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 1451 Figure 3. Accurate determination of the dissociation constant Kd by using the force-spectroscopy-based single-molecule binding assay. The fractions of Ni2 +-bound G6-53 (Y) are plotted against the total concentration of Ni2+ in the binding isotherm. Black symbols were measured using a pulling speed of 400 nms1, whereas grey symbols represent the results from AFM experi- ments using a pulling speed of 2000 nms1. The solid line is the fit to the binding isotherm using Equation (2), which is based on a single-binding-site model and takes into account all the species present in solution.

To compare the Kd values measured by using single-mole- cule force spectroscopy with those obtained from traditional ensemble methods, we also carried out ensemble chemical un-

folding studies of G6-53 to determine Kd. This method is based on measurements of the changes in equilibrium stability (DDG) upon addition of metal ions and has been widely ap- plied to study the binding of metal ions to proteins.[17,18] The Figure 2. Unfolding force histograms of G6-53 in the absence and presence 2+ of different concentrations of Ni2 +. The unfolding force histograms of apo- relationship between DDG and Kd and [M ], which can be de- G6-53 (e.g. 0mm Ni2+) and G6-53 in the presence of a saturating concentra- rived based on a thermal dynamic cycle analysis,[18] obeys the tion of Ni2 + (4mm Ni2+) are unimodal distributions with average values at following equation [Eq. (1)]: 120 and 250 pN, respectively. In contrast, the unfolding force histograms of G6-53 in the presence of a non-saturating concentration of Ni2 + show 2þ 2þ two clear separate peaks: one is found at 120 pN, corresponding to the KMN þ½M KMD þ½M 2 + DDG ¼ RT ln RT ln ð1Þ unfolding of Ni -free G6-53, and the other is found at 250 pN, corre- KMN KMD sponding to the unfolding of Ni2 +-bound G6-53. The initial concentration of Ni2 + for each histogram is shown on the right. The solid lines are Gaussian where DDG is the difference in thermodynamic stability of the fits to the experimental data. protein in the presence and absence of metal ions, R is the gas

constant, and T is the absolute temperature. KMN and KMD denote the dissociation constants for the binding of metal ions unfold at higher forces. Thus, we can directly determine the to proteins in the native and denatured states, respectively. To fraction of Ni2+-bound G6-53, Y, at each metal-ion concentra- derive Equation (1), the binding of metal ions to proteins in tion and plot the binding isotherm (Figure 3). We fitted the both native and unfolded states is assumed to follow a 1:1 sto- binding isotherm to a single-site binding model, which takes ichiometry and the folding/unfolding of proteins is assumed to into account all the species present, and measured a dissocia- follow a two-state model. These assumptions, which cannot be 2+ tion constant Kd of 98 mm for the binding of Ni to G6-53. fully justified in many cases, are necessary to extract the bind- It should be noted that the partition of G6-53 domains be- ing constant from the experimental data. tween the apo and Ni2+ bound-forms at a given Ni2+ concen- The thermodynamic stability of G6-53 in the presence of dif- tration is solely dependent upon the proteins’ intrinsic binding ferent concentrations of Ni2+ was measured through GdmCl equilibrium with Ni2+ and will not be affected by the experi- denaturation, as shown in Figure 4A. Changes in the thermo- mental parameters of the AFM studies. Therefore, although the dynamic stability of G6-53 upon binding of Ni2+ were plotted mechanical stability of both forms of G6-53 can be influenced against the concentration of Ni2+ added to the system, as by the pulling speeds, the partition of G6-53 between the two shown in Figure 4B. We fitted the data to Equation (1) and m forms should remain unaffected. To illustrate this point, we car- measured a dissociation constant Kd of 260m for the binding ried out force spectroscopy measurements at two different of Ni2+ to G6-53. This value is similar to that measured by pulling speeds (i.e. 400 and 2000 nms1). As shown in Figure 3, using single-molecule force spectroscopy. the binding curves measured at these two different pulling Since the mechanical stability of proteins is a kinetic stabili- speeds are indistinguishable from each other. ty,[19] single-molecule AFM experiments essentially measure the

1452 www.chemphyschem.org 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2009, 10, 1450 – 1454 of the IgG was much higher than that measured by using co-localization-based surface plasmon resonance spec- troscopy. Traditional metal-binding assays have been—and will con- tinueACHTUNGRE to be—the dominant methods of choice for determining metal-binding affinity to proteins. Such ensemble methods re- quire large amounts of proteins and are often time consum- ing.[9] In special cases (for example, when the proteins cannot be easily obtained in large quantities), the traditional ensemble methods may become inadequate. In addition, assumptions used in some assays cannot be fully justified in many cases and may bias the interpretation of the experimental data. For example, the assumption of a 1:1 stoichiometry between the unfolded protein and the metal ion in the thermodynamic method shown above is not necessarily accurate, as the two histidine residues may bind Ni2+ in an independent fashion. Moreover, if the folding/unfolding of metal-binding proteins is more complicated than a two-state process, the thermal dena-

turation method can no longer be used to determine Kd,be- cause Equation (1) is not justified. Therefore, developing new and complementary metal-binding assays will be of particular interest. Our single-molecule force-spectroscopy-based metal- binding assay is one of such efforts. Compared with traditional ensemble binding assays, the force-spectroscopy-based method reported herein is a conceptually simple and yet pow- erful single-molecule technique. The direct measurement of the partition of proteins between the apo and metal-bound Figure 4. Measuring the dissociation constant Kd via the equilibrium stability forms circumvent certain uncertainties encountered in ensem- method: A) Standard GdmCl denaturation curves of G6-53 in the presence of different concentrations of Ni2 +. The solid lines are fits to the experimen- ble binding assays and enables the direct determination of Kd tal data obtained by using Equation (3). B) Changes in the thermodynamic without invoking any assumption. Another advantage is the stability of G6-53 upon addition of Ni2 +. A fit (solid line) to the experimental minimum amount of proteins required in the force-spectrosco- data, obtained by using Equation (1), gives a dissociation constant Kd of py-based metal-binding assay. For example, the amount of 260mm for the binding of Ni2 + to G6-53. G6-53 used in this case is a thousand times less than that used in chemical denaturation based assays. Furthermore, this kinetic consequence of the binding of Ni2+ to proteins. In con- method is a single-molecule measurement in nature and is ca- trast, chemical denaturation experiments measure the effects pable of detecting any heterogeneity and binding intermedi- of metal-ion binding on the thermodynamic stability of pro- ate in the binding system, and hence, it can potentially reveal teins. Despite the differences between these two methods, details of the binding mechanism that are otherwise difficult both are functional binding assays and both report the ther- to obtain. In particular, this method can be extended to the modynamic consequence of the binding of Ni2+ to G6-53. This study of other complex metal-binding systems, such as RNA common feature between the two different binding assays and DNA apatmers, where metal binding is critical for the fold- [1] readily explains why the value of Kd measured by using force ing and biological functions of these systems. Recent single- spectroscopy is close to that measured for free G6-53 in solu- molecule force spectroscopy studies revealed that binding of tion by using the chemical denaturation method. metal ions can generate specific mechanical signatures.[20,21] However, it is important to note that the excellent agree- Therefore, we anticipate that the novel force-spectroscopy- ment between the Kd value measured by using force spectro- based metal-binding assay developed herein will complement scopy and those obtained with other ensemble methods the existing ensemble metal binding assays and find unique cannot be generalized to other binding systems, such as pro- applications in a range of complex systems in chemistry and tein–protein interactions. For protein–protein interactions, Kd is life sciences, where conventional methods are inadequate or frequently measured using co-localization-based binding difficult to apply. assays, which are susceptible to false positives and tend to Despite these unique features, the single-molecule force- overestimate the binding affinity. In such systems, the Kd value spectroscopy-based metal-binding assay has its own limita- obtained from force spectroscopy may be different from those tions. Similar to any other metal-binding assay, this new bind- measured using co-localization-based methods. Our previous ing assay is not universally applicable. Since it is based on the studies[13] on the binding of a small protein, GB1, to the Fc difference in mechanical stability between the apo and bound fragment of IgG showed that the Kd value measured by using forms, this method is not applicable to metal-binding systems force spectroscopy for the binding of GB1 to the Fc fragment in which the metal binding does not change the mechanical

ChemPhysChem 2009, 10, 1450 – 1454 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 1453 stability of the proteins. Thus, the applicability of this method free energy of unfolding in the absence of denaturant, R is the gas will not be as general as that of thermal-denaturation-based constant, and T is the absolute temperature in Kelvin. binding assays. Moreover, the resolution of this binding assay is also limited by the relatively low force resolution of the AFM technique. Currently, it is not feasible to apply the force-spec- Acknowledgements troscopy-based metal-binding assay to determine the Kd value for the binding of metal ions to proteins with unfolding forces This work was supported by the Natural Sciences and Engineer- below 20 pN. Further improvements in the sensitivity of force ing Research Council of Canada, Canada Foundation of Innova- measurements with AFM, as those recently reported by Rief tion and Canada Research Chair program. H. L. is a Michael [22] and co-workers, will be required to expand the applicability Smith Foundation for Health Research Career Inverstigator. of this new binding assay to a much broader range of systems.

Keywords: binding assay · cations · force spectroscopy · Experimental Section proteins · single-molecule studies ACHTUNGRE [14] (G6–53)8 was engineered as described previously. The polypro- 2+ tein was expressed in DH5a strain, purified by Co affinity chro- [1] S. J. Lippard, J. M. Berg, Principles of Bioinorganic Chemistry, University m matography, and eluted in PBS buffer with 300m NaCl and Science Books, Mill Valley, 1994. 150 mm imidazole. EDTA (20mm) was added to the elution frac- [2] J. J. R. F. da Silva, R. J. P. Williams, The Biological Chemistry of the Ele- tions to remove any residual Co2+ that may exist in the eluted frac- ments: The Inorganic Chemistry of Life, 2nd ed., Oxford University Press, tion. The proteins were further dialyzed against Tris-HCl buffer Oxford, 2001. (10mm, pH 7.4, containing 100mm NaCl) to completely remove [3] I. Bertini, A. Sigel, H. Sigel, Handbook on metalloproteins, Marcel Dekker, EDTA and imidazole. New York, 2001. [4] D. W. Christianson, J. D. Cox, Annu Rev Biochem 1999, 68, 33. Single-molecule AFM experiments were carried out on a custom- [5] R. H. Holm, P. Kennepohl, E. I. Solomon, Chem. Rev. 1996, 96, 2239. built AFM, as described previously.[23,24] All the force-extension [6] T. Dudev, C. Lim, Annu Rev Biophys 2008, 37, 97. measurements were carried out in Tris-HCl buffer (10mm, pH 7.4, [7] C. J. Wilson, D. Apiyo, P. Wittung-Stafshede, Q Rev Biophys 2004, 37, m 285. containing 100m NaCl) with different concentrations of NiCl2. The spring constant of the AFM cantilevers (Si N cantilevers from [8] K. A. Connors, Binding constants : the measurement of molecular com- 3 4 plex stability, Wiley, New York, 1987. Veeco) was calibrated before each experiment by using the equi- 1 2+ [9] E. Freire, W. W. van Osdol, O. L. Mayorga, J. M. Sanchez-Ruiz, Annu Rev partition theorem and a typical value was 60 pNnm . For the Ni Biophys Biophys Chem. 1990, 19, 159. 2+ -binding studies, we first deposited the polyprotein and the Ni [10] X. Michalet, S. Weiss, M. Jager, Chem. Rev. 2006, 106, 1785. solution onto a glass coverslip containing 50 mL of Tris-HCl buffer [11] H. D. Kim, G. U. Nienhaus, T. Ha, J. W. Orr, J. R. Williamson, S. Chu, Proc. and mixed them in situ. The AFM experiments were carried out Natl. Acad. Sci. USA 2002, 99, 4284. after allowing the mixture to equilibrate for about 30 min. Unless [12] T. Ha, X. Zhuang, H. D. Kim, J. W. Orr, J. R. Williamson, S. Chu, Proc. Natl. otherwise noted, the pulling speed was 400 nms1 for all the AFM Acad. Sci USA 1999, 96, 9077. experiments. [13] Y. Cao, M. M. Balamurali, D. Sharma, H. Li, Proc. Natl. Acad. Sci. USA 2007, 104, 15677. The binding curve obtained from single-molecule force spectrosco- [14] Y. Cao, T. Yoo, H. Li, Proc. Natl. Acad. Sci. USA 2008, 105, 11152. py data was fitted using by using Equation (2) to determine Kd for [15] M. Rief, M. Gautel, F. Oesterhelt, J. M. Fernandez, H. E. Gaub, Science the binding of Ni2+ to G6–53: 1997, 276, 1109. [16] M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P. E. Marszalek, S. E. Broedel, J. Clarke, J. M. Fernan- ½G6 53 þ½Ni2þ þ K ð½G6 53 þ½Ni2þ þ K Þ2 4½G6 53 ½Ni2þ Y ¼ 0 0 d 0 0 d 0 0 dez, Proc. Natl. Acad. Sci. USA 1999, 96, 3694. 2½G6 530 [17] F. H. Arnold, B. L. Haymore, Science 1991, 252, 1796. ð2Þ [18] B. A. Krantz, T. R. Sosnick, Nat. Struct. Biol. 2001, 8, 1042. [19] H. Li, A. F. Oberhauser, S. B. Fowler, J. Clarke, J. M. Fernandez, Proc. Natl. where [G6–53]0 is the initial equivalent concentration of G6–53 do- 2+ 2+ Acad. Sci. USA 2000, 97, 6527. mains in solution, [Ni ]0 is the total concentration of Ni in solu- tion, and K is the dissociation constant.[25] [20] J. Liphardt, B. Onoa, S. B. Smith, I. J. Tinoco, C. Bustamante, Science d 2001, 292, 733. Chemical denaturation experiments were carried out on a Cary [21] B. Onoa, S. Dumont, J. Liphardt, S. B. Smith, I. Tinoco, Jr., C. Bustamante, Eclipse Fluorescence Spectrophotometer. Tryptophan fluorescence Science 2003, 299, 1892. of G6–53 mutant was excited at 280 nm, and the emission was [22] J. P. Junker, F. Ziegler, M. Rief, Science 2009, 323, 633. monitored at 350 nm to probe the unfolding process. The fluores- [23] Y. Cao, C. Lam, M. Wang, H. Li, Angew. Chem. Int. Ed. 2006, 45, 642; Angew. Chem. 2006, 118, 658. cence data were normalized according to standard procedures and [26] [24] Y. Cao, H. Li, Nat. Mater. 2007, 6, 109. fitted to Equation (3): [25] I. Segel, Enzyme Kinetics, John Wiley, New York, 1975. [26] A. Fersht, Structure and mechanism in protein science: a guide to enzyme exp½ðm ½DDGH2O Þ=RT F ¼ DN ð3Þ catalysis and protein folding, Freeman, New York, 1999. H2O 1 þ exp½ðm ½DDGDNÞ=RT [27] J. F. Marko, E. D. Siggia, Macromolecules 1995, 28, 8759. where F is the fraction of unfolded proteins, m is the slope of the Received: April 28, 2009 H2O transition, [D] is the concentration of the denaturant, DGDN is the Published online on June 9, 2009

1454 www.chemphyschem.org 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2009, 10, 1450 – 1454