15910
J. Phys. Chem. B 2004, 108, 15910-15918
Probing Structural Dynamics of Individual Calmodulin:Peptide Complexes in Hydrogels by Single-Molecule Confocal Microscopy Jianyong Tang,† Erwen Mei,† Clive Green,† Justin Kaplan,‡ William F. DeGrado,†,‡ Amos B. Smith, III,† and Robin M. Hochstrasser*,† Department of Chemistry and Department of Biophysics and Biochemistry, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104 ReceiVed: May 4, 2004; In Final Form: July 19, 2004
An environment-sensitive probe, Nile Red, tagged to a designed model peptide, is used to probe the structural changes and dynamics of single calmodulin (CaM):peptide complexes. When the Ca2+ concentration changes from 0 M to 5 mM, the labeled Nile Red dye undergoes an order of magnitude increase in fluorescence intensity. By encapsulation of single CaM:peptide-Nile Red complexes into a hydrogel, single-molecule fluorescence detection has been achieved using confocal microcopy. The fluorescence polarization distribution obtained for these single complexes shows a mean of 0 and a width of 0.17, for a binning time of 1 ms. This finding shows that on the 10-100-µs time scale the CaM:peptide complex is tumbling within the hydrogel matrix. The single-molecule spectral distribution provides the scale of the heterogeneity of the polarity sensed by the probe. The calcium concentration dependency of the single-molecule-fluorescence lifetime distributions and photon-arrival-time (PAT) trajectories of the CaM:peptide-Nile Red complexes were also obtained. The mean and variance of the Nile Red fluorescence decay rate increase 40% and 180%, respectively, as the Ca2+ concentration approaches the titration midpoint of 2 µM. These changes are considerably greater than would be expected if the chromophore was in a homogeneous static environment, or in a heterogeneous environment that was exchanging faster than a time scale of 1 s to 10’s of seconds. Thus, PAT analysis appears to be uniquely well suited to study the dynamics of the peptide dissociation from the CaM:peptide complex in the presence of [Ca2+] and transitions within heterogeneous populations on the microsecond-second time scale.
Introduction Single-molecule technology permits signal detection from individual molecules or molecular complexes.1-9 Thus, the real distribution of molecular and dynamic properties can be observed directly, without a need for synchronization. Such information cannot be obtained via bulk measurements; bulk signals are an accumulation of signals from all molecules, and the individual molecule information is lost during the averaging process. In a homogeneous system, such information defines just the statistical uncertainty, which can be deduced from theory or simulation. However, for a heterogeneous system such as a protein, this information is crucial to understanding the structure and dynamics of the molecular system under investigation. This is because the measurement of underlying population distributions brings new information, such as multimodal conformations and the diversity of reaction pathways. In this paper, a molecular system consisting of the protein complex Calmodulin:peptide10,11 has been studied by singlemolecule approaches. Recently, Allen et al.12 have reported single-molecule experiments on calmodulin using a quite different approach.12 Calmodulin (CaM) is a regulatory protein involved in a variety of cellular calcium-dependent signaling pathways.13-15 With just 148 residues, calmodulin, upon binding four calcium ions, undergoes conformational changes in its role of modulation of the activities of a large number of proteins (including protein kinases, NAD kinase, phospho-diesterase, and †
Department of Chemistry, University of Pennsylvania. Department of Biophysics and Biochemistry, University of Pennsylvania. ‡
Figure 1. Synthesized Nile Red dye with amine-reactive linker.
calcium pumps, as well as proteins involved in motility). For this work, a peptide was designed that is intended to deliver an environment-sensitive fluorophore, Nile Red (Figure 1), to the apolar binding site of CaM. CaM can assume a number of different conformations by virtue of a flexible linker connecting two globular domains responsible for binding calcium ions and target enzymes.14,16,17 CaM recognizes a basic amphiphilic R-helix in a large number of the targets, and CaM engulfs the substrate between the two domains. These peptides often contain aromatic residues, which become sequestered in hydrophobic invaginations within the binding sites. Previously, a model CaMbinding helix (sequence KKLLKLLKKLLKL-carboxamide) was prepared, and it was shown to bind CaM with nM affinity.11 Aromatic groups (fluorenyl, indole, or benzophenone)18 attached to the N-terminus of the peptide were shown to increase the
10.1021/jp0480798 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/14/2004
Individual Calmodulin:Peptide Complexes affinity of the peptide for CaM, and to locate at the C-terminal lobe of the CaM structure.11 Sequence analysis and photoaffinity labeling indicated that such peptides interact with CaM in a manner analogous to smooth-muscle myosin light-chain kinase (MLCK), for which there is a high-resolution crystal structure. The degree of burial of the aromatic group near the end of the peptide is of particular relevance to this work. In the structure of MLCK,18-20 a Trp occupies an analogous position, and the side chain is essentially fully buried in the C-terminal lobe of CaM. However, modeling indicated that this pocket is not sufficiently large to accommodate the full Nile Red ring structure, so the fluorophore could be partially exposed to solvent. Indeed, in the structure of a complex of CaM with the tricyclic drug trifluoperazine, multiple drug molecules are located at the C-terminal lobe and are partially exposed to solvent.21 Thus, a key expectation for this work is that the Nile Red dye will adopt a partially solvated, and possibly also heterogeneous environment. Single-molecule spectroscopy should therefore provide an excellent approach for studying the dynamics of the Nile Red peptide bound to CaM. Nile Red is a highly sensitive probe for hydrophobicity and polarity.22 In solvents with various polarities, Nile Red shows very different absorptions, fluorescence, fluorescence lifetimes, and fluorescence quantum yields. The spectral sensitivity of Nile Red to solvent polarity is attributed to a twist-internal-charge transfer (TICT) involving motions of the diethylamino group.23,24 The fluorescent spectrum and lifetime of Nile Red therefore represent a molecular-scale probe of local environments in individual protein pockets, and Nile Red is expected to reveal the structural and dynamic heterogeneity of the CaM:peptide complexes. We will use single-molecule photon arrival time (PAT) trajectories25,26 to monitor the dynamic process of single CaM:peptide complexes. PAT trajectories record the fluorescent photons from each molecule on a photon-by-photon basis. Each photon is described by two parameters: a macrotime, which is the time between this detected photon and the starting time of measurement, and a microtime, which is the time between this detected photon and the laser pulse that excites the molecule to the electronic excited state. The macrotime series reflect the photon density distributed in time, and they can be transformed into conventional intensity trajectories. However, these series cannot be obtained from intensity trajectories where the photon-byphoton information is lost. The microtime series reflect the molecular status at the time of each emitted photon because each value of the microtime is a realization of the instantaneous fluorescence lifetime convoluted with the instrumental response function. The histogram of a sufficiently long enough microtime series provides the conventional time-correlated-single-photoncounting (TCSPC) fluorescence decay curve. Just as with the transformation from the macrotime series to intensity trajectories, the transformation from the microtime series to a conventional lifetime is also not a process that can be inverted, because the histogram of microtimes does not have the sequential information of the photons in the bins. So, PAT trajectories are expected to give more information than conventional intensity trajectories combined with TCSPC measurements. Moreover, since the time intervals between detected photons are stochastically distributed around the mean interval, which is the inverse of photon count rate, very fast dynamic processes can be resolved by PAT analysis. Furthermore, the intrinsic limited survival time of single chromophores is more effectively used in a PAT analysis, which extracts information from each fluorescent photon.
J. Phys. Chem. B, Vol. 108, No. 40, 2004 15911 As a method of bioencapsulation, the sol-gel technique has been widely employed in the field of biosensors.27,28 Enzymes and other proteins that bind substrates, inhibitors, or other related molecules can be immobilized with this transparent matrix that stabilizes the proteins and does not block the substrate molecules from moving in or out of the matrix. The response of the proteins can then be observed by optical measurement. In this paper, a hydrogel (a predried solgel) has been used to immobilize single CaM:peptide complexes. The highly transparent hydrogel glass film is ideally suitable for fluorescence confocal microscopy. Materials Nile Red dye tag possessing an amine-reactive linker (Figure 1) was synthesized according to the method of Briggs et al.29 CaM from bovine brain with a purity of >95% was purchased from Sigma (P-2277). As described in a previous work,10 a peptide designed to bind tightly to the calmodulin binding pocket, Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-LeuLeu-Lys-Leu-amide, was synthesized (0.25-mM scale) using an Applied Biosystems 433A solid-phase peptide synthesizer (Perkin-Elmer), N-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids, and 2,4-dimethoxybenzhydrylamine resin. Standard coupling conditions using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxybenzotriazole hydrate (HOBt), diisopropylethylamine (DIEA) in N-methylpyrrolidinone (NMP), and a 4-fold molar excess of amino acid were used for all couplings. The N-terminus was labeled with the Nile Red tag by stirring resin-bound peptide in dimethylformamide (DMF) with an approximately 4-fold molar excess of Nile Red tag with amine reactive linker, HOBt, and HBTU, and a 6-fold molar excess of DIEA; identical aliquots of HOBt, HBTU, and DIEA were added again after 6 h, and the reaction mix was stirred overnight. The resin was washed first with DMF and then with ether. It was then dried; the peptide was removed from the resin via treatment for 1.5 h in 95% TFA and 5% water, precipitated with cold ether, solubilized in water, and lyophilized. The crude peptide was purified at room temperature by reverse-phase HPLC using a semipreparative C18 column (Vydac) and a linear gradient (3 mL/min) of water with 0.1% TFA and an acetonitrile/water/ TFA mixture (90/10/0.1). Analytical HPLC and MALDI-TOF mass spectrometry of this purified peptide verified the homogeneity and identity. In experiments, the tagged peptide and CaM were dissolved into 50 mM Tris buffer at pH 7.0 with 150 mM NaCl. To control the calcium-ion concentration at a low level, a calcium buffer kit (in 30 mM MOPS buffer, pKa ) 7.2) from Molecular Probes (C3009) was used. In bulk experiments, the CaM concentration was ∼10-7 M and in single-molecule experiments the CaM concentration was ∼10-9 M. Tetramethoxysilane (TMOS) was purchased from Aldrich (99+%). Methods Hydrogel Immobilization. Following a protocol used by others,30 TMOS (900 µL) is mixed with 200 µL water and 8 µL 0.5 M HCl, then the mixture is immediately put into an ice bath and sonicated for 30 min. The resulting sol solution is maintained in the ice bath afterward. To make thin films, 100 µL sol is mixed with 100 µL Tris buffer (50 mM; pH ) 7.0, with 150 mM NaCl) solution and stirred well. Then, 50 µL of the solution with calcium:calmodulin:peptide (ca. 10-8 M) is added to this mixture. Upon adding buffer, the acidic condition
15912 J. Phys. Chem. B, Vol. 108, No. 40, 2004 is changed to near pH ) 7, which accelerates gel formation. About 2 µL of this mixture is quickly transferred onto a clean cover-glass surface and another clean cover glass is applied to make a sandwich. Within 2 min after the transfer, a highly transparent glasslike film is formed, which is ready to be put onto the confocal microscope stage for single-molecule experiments. The encapsulant is a hydrogel; when dried, it would become a conventional solgel after contraction by a factor of ca. 5.27,31 All of the single-molecule works described here were carried out in hydrogels. Experimental Instrumentation. The bulk optical absorption spectra were acquired using a Perkin-Elmer UV/Vis Spectrometer Lambda Bio 40. The single-molecule fluorescence detection was achieved by confocal microscopy using a home-built inverted scanning confocal microscope or a confocal microscope built based on a commercial Nikon DIAPHOT 300 inverted microscope, as described previously.2,32 The single molecules were excited by a 76-MHz mode-locked Nd:YAG laser (Coherent Antares) frequency doubled to 532 nm with incident laser power of 0.5-2 µW. The count rates from a typical single fluorescent molecule are around 2000-5000 counts/s. The excitation light was circularly polarized. One or two silicon avalanche photodiodes (APD) from EG&G (now Perkin-Elmer Optoelectronics) were employed for one-channel (fluorescence lifetime) or two-channel (fluorescence polarization) singlemolecule detection, respectively. For single-molecule polarization measurements, a broadband polarizing cube beamsplitter (Newport) was used to guide the s and p polarized fluorescence to two APDs, permitting measurement of the projection of the emitting dipole onto two perpendicular directions in the focal plane. A dichroic mirror (DCLP 550, Chroma) was employed to separate the laser and fluorescence paths, and a set of filters (a 532-nm notch filter from Kaiser, a long-pass filter 590 LP from SCHOTT, and a 630 nm/60 nm band-pass filter from Chroma) was used to remove backscattering of the laser and impurity fluorescence. To measure single-molecule spectra, a nitrogen-cooled CCD camera system (Princeton Instruments, now Roper Scientific) coupled with a monochromator was employed. The typical data acquisition time in a single-molecule fluorescence spectral measurement is 30 s. This setup was also used for the bulk calcium-ion titration measurement. For fluorescence-lifetime measurements of bulk samples, a Nd:YAG excitation laser synchronously pumped a dye laser emitting at 552 nm with a repetition rate of 3.8 MHz, which was employed. The TCSPC instrument response function was ∼80 ps. The single-molecule fluorescence lifetimes were measured by an integrated multiplex TCSPC board (Becker&Hickl GmbH, Germany), using a frequency-doubled Nd:YAG excitation laser. The instrumental-response function of 250 ps was limited by the time response of the APD and the laser pulse width. For rapid acquisition of fluorescence lifetime data for the immobilized single molecules, the lifetime imaging technology was used. This was realized by using the Becker&Hickl SPC-730 board synchronized with a scanning stage controller (Digital Instruments, Nanoscope IIIa). In each fluorescence lifetime image, the fluorescence decay curves were measured and stored on a pixel-by-pixel basis. Groups of pixels around each singlemolecule area were binned before the lifetime fitting procedure was applied. The equivalent binning time for each singlemolecule lifetime measurement is around several seconds. Photon Arrival Time (PAT) Trajectory. Each PAT trajectory was obtained using single-photon-counting hardware (Becker&Hickl SPC-630). A typical raw data file contains a
Tang et al. fluorescence photon series, with the macrotime and microtime being recorded for each detected photon. To characterize the lifetime fluctuation from the PAT trajectory, a previously reported protocol25,33 was used. In this protocol, the lifetime autocorrelation function C2(t) is defined as
C2(t) ) 〈δγ(t)-1δγ(0)-1〉 ) 〈δγ-2〉 C /2(t) C /2(t) )
〈γ(t)-1γ(0)-1〉 - 〈γ-1〉2 lim 〈γ(t)-1γ(0)-1〉 - 〈γ-1〉2 tf0
where γ(t) is the fluorescence decay rate at time t, and δγ(t)-1 ) γ(t)-1 - 〈γ-1〉. To calculate C2(t) from the microtime and macrotime series {τp, tp} (p is the photon index), the following formulas are used:26,33
〈γ(t)-z〉 )
〈γ(t)
-z1
-z2
γ(0)
〉)
1
1 TA0
τpz-1
∑ Γ(z) {p}
∑
τqz1-1
(T - t)A02∆ {q > p},{p} Γ(z1)
u(tq - tp τpz2-1 t) Γ(z2)
where T is the total length of the single-molecule trajectory, A0 ) hI/γ-1 is the proportionality constant that compensates for the lifetime-dependent fluorescence intensity I, Γ(z) is the gamma function, and ∆ is the preset time bin that defines the time resolution; u(t) is defined as
1 1 u(t) ) 1 for - ∆ e t e ∆ and u(t) ) 0 otherwise. 2 2 In the formula above, if we confine the computation to the first-order two-time correlations in C2(t), the microtime does not contribute. This arises because of the assumption that fluorescence lifetime is proportional to the fluorescence intensity. In that case, the C2(t) is essentially an intensity fluctuation. However, this generalized formula enables the calculation of higher-order correlation functions; in future work, these could be more informative for the single-molecule dynamics. In this paper, we consider only first-order correlations. To calculate the overall correlation curve for a number of single molecules, all the lifetime autocorrelation functions obtained by the protocol described above for a group of molecules were summed and divided by the standard deviation of this summed correlation curve. Data Analysis. Data collected from these experiments were analyzed by the software packages IGOR (Wavemetrics) and Matlab (Mathworks, R12). The software package SPCImage 2.0 (Becker&Hickl) was used to analyze the fluorescence imaging results with least-squares fitting. Results and Discussion Nile Red Peptide. The Nile Red peptide was synthesized via a solid-phase method, similar to previously described derivatives.10 Nile Red interacts with hydrophobic surfaces of some proteins with an apparent dissociation constant in the range of 0.6 µM,22 while the synthesized CaM:peptide has a Kd )
Individual Calmodulin:Peptide Complexes
J. Phys. Chem. B, Vol. 108, No. 40, 2004 15913
Figure 2. Bulk measurement of fluorescence spectra of CaM:peptideNile Red complexes. Excitation laser wavelength: 532 nm; top curve: with 39 µM [Ca2+]; bottom curve: with 0 µM [Ca2+].
Figure 3. Fluorescence decay measurement of CaM:peptide-Nile Red complexes. Dashed line: sample fluorescence decay; solid line: instrumental response function.
0.2 nM11-5 pM.34 So, we assumed that the Nile Red does not alter the affinity of the peptide and CaM. Also, the charges of the Lys residues, as well as the nonpolar Leu, cause the peptide to bind to CaM more tightly. Moreover, fluorescence-anisotropy measurements showed that the anisotropy decay time of the CaM:peptide-Nile Red complexes in buffer solution is around 7 ns, which is consistent with the expectation of the rotational correlation time of CaM under the same experimental conditions, but is much slower than the rotational correlation time of free Nile Red dye in a solution ( ∼100 µM). Also, given the expected range of dissociation constants and a near-diffusion controlled rate of association (∼107 M-1 sec-1), we estimate that the rate of dissociation of the peptide from the protein will be on the order of 10-2-10-4 sec-1. Thus, during the time of observation of a single molecule (from seconds to 10’s of seconds), the complex is unlikely to dissociate. Any conformational averaging that is observed reflects motions that do not require a full dissociation of the peptide. Bulk Absorption and Fluorescence Spectroscopy. The bulk fluorescence properties of the Nile-Red-labeled peptide were first measured in the presence of apo- and calcium-loaded CaM. The binding of calcium to CaM induces the formation of hydrophobic binding sites within the two lobes. These sites should bind to the Nile Red group, inducing a large increase in the quantum yield. The bulk absorption spectra from the NileRed-labeled peptide in solution or the presence of calciumsaturated CaM both display absorption peaks around 550 nm. The fluorescence spectra for the CaM:peptide-Nile Red in the presence of saturating 39 µM Ca2+, and in the absence of added Ca2+, are shown in Figure 2. In the absence of Ca2+, the spectrum was barely detected above the background signal, which includes a significant contribution from the Raman scattering from water. The Nile Red-peptide fluorescence in solution (not shown in Figure 2) increased ca. 10 times after adding apo-CaM; addition of Ca2+ causes a 20-fold further
increase in quantum yield. The finding of a small increase in quantum yield, even in the absence of added calcium ions, might be a result of a low level of calcium impurity in the buffer. Alternatively, the peptide is expected to bind with significantly lower affinity to CaM in a calcium-independent manner. Interestingly, though the fluorescence intensity varies as a function of calcium-ion concentration, the fluorescence peak positions are not significantly different for the CaM:peptide samples at high and at very low calcium-ion concentrations. Thus, if the peptide binds in a calcium-independent manner, it is possible that the apo-CaM and calcium-ion-loaded CaM adopt similar conformations upon binding to the target peptide. The fluorescence spectrum of Nile Red in the CaM:peptide complex is nearly identical to that of Nile Red in methanol,35 suggesting that the polarity of the protein-binding pocket of CaM roughly matches that of methanol solution. These data clearly show the utility of the Nile Red probe for evaluating the environment of the peptide on the surface of CaM. Bulk and Single-Molecule Fluorescence Lifetime Measurement. The fluorescence lifetime measured from the bulk solution of CaM:peptide-Nile Red complex (5 mM CaCl2, 50 mM Tris buffer, pH ) 7.0, and 150 mM NaCl) by conventional TCSPC is shown in Figure 3. The deconvolution of this fluorescence-decay curve shows nonexponential behavior consistent with two lifetime components of 0.69 and 4.2 ns (κ2 ) 1.10), with weights of 57% and 43%, respectively. The time scale of the interchange of the structures contributing to this nonexponential decay is not known. Fluorescence-lifetime measurements of bulk solutions by TCSPC using the confocal microscope instead of a conventional sample cell yielded an additional short lifetime component, which was shown to be a result of significant (∼3%) light scattering. This scattering was also present when the hydrogel was used as a solvent. Neglecting this scattering component, the fluorescence-decay function in the hydrogel was not significantly different from that found in solution. An interpretation of the small differences between the bulk lifetime data and the single-molecule accumulated lifetime data is not considered meaningful because the excitation lasers, detectors, and instrumental response functions are different in these two experimental configurations, as described in the instrumentation section. When the time-resolved fluorescence signals from all the single molecules were added up, the overall fluorescence decay showed the strong scattered-light component in addition to 509-ps (27%) and 3.3-ns (73%) components. The reason the short lifetime components are not present in the single-molecule lifetime distribution is that in the data analysis an exponential decay from each molecule was assumed. The single-molecule fluorescence data do not have enough counts
15914 J. Phys. Chem. B, Vol. 108, No. 40, 2004
Figure 4. Calcium-ion titration measurement of CaM:peptide-Nile Red complexes. Solid line: the hyperbolic fitting curve.
per molecule to establish whether they are multi-exponentials, so a single-exponential fitting procedure needed to be used. Cser et al.23 found that the fluorescence lifetime of Nile Red is related to the hydrogen-bonding strength between a Nile Red molecule and the local environment. In addition, the local polarity (scaled by ET(30)) has approximately linear dependence of the Nile Red fluorescence lifetime in solvents capable of forming hydrogen bonds. This hydrogen bond property can be scaled by Abraham’s ΣR2H.36 Calcium Titration Curve. The fluorescence from CaM:peptide-Nile Red complexes at various calcium concentrations was analyzed by a normalization formula:
Rf )
F - Fmin Fmax - Fmin
In this equation, Rf is the relative fluorescence, F is the observed fluorescence intensity; Fmin and Fmax are the minimum and maximum fluorescence levels. The measured titration curve is shown in Figure 4 under the condition that the concentrations of CaM and Nile-Red-labeled peptide are both 10-7 M. A hyperbolic fit of the curve indicates the midpoint is at 2 µM Ca2+. This is close to the calcium-binding curve obtained by measuring cyclic nucleotide phosphodiesterase enzyme activity, which is Ca 2+:CaM dependent.37,38 The calcium titration also provides information that can be used to manipulate the experimental conditions to study CaM:peptide complex dynamics at different calcium levels. At very high Ca2+, a more homogeneous ensemble consisting of the protein with one peptide and four calcium ions would predominate. In contrast, near the midpoint, the most heterogeneous population will be observed. Under this condition, one expects a combination of species with partially occupied Ca2+-binding sites, which, in turn, will result in partial exposure of the hydrophobic surfaces. Very extensive experiments in aqueous solution (e.g., Ca2+-binding isotherms at different CaM:peptide concentrations and peptide-binding isotherms at various Ca2+ concentrations) would be necessary to probe the full extent of the heterogeneous species existing near the midpoint of the calcium titration curves. Many important bulk experiments targeting the calcium-cooperative binding of CaM have been reported.39-41 On the other hand, single-molecule spectroscopy is more suited to measuring the extent of heterogeneity of the distribution, as well as the rates of interconversion of individual members of the ensemble from the nanosecond to the second time scale. Single-Molecule Fluorescence Polarization Measurement. A number of experiments were conducted to ensure that the CaM:peptide complexes are efficiently encapsulated within a
Tang et al. fluid environment within the hydrogel matrix. The Nile-Redlabeled peptide was first added to the hydrogel, with or without calcium, under the same experimental conditions as used for the protein experiments. The single-molecule fluorescence of Nile Red was found to be too weak to be detected. Thus, we assumed that any detectable single-molecule fluorescence in the hydrogel is exclusively from the CaM:peptide-Nile Red complexes. Then, the effect of the matrix on the fluorescence of single CaM:peptide-Nile Red complexes at high calcium concentration was measured. The fluorescent photons from single CaM:peptide-Nile Red complexes in the hydrogel matrix are found to have spectra that are the same within experimental errors as for the complex in buffer solution. The fluorescence lifetime is also the same as in buffer solution. Because the fluorescence of Nile Red is sensitive to the CaM:peptide complex conformation, the structure of this complex is considered unchanged by the hydrogel microenvironment. In another experiment, the hydrogel effect on overall motions of complexes was measured. In these experiments, each individual molecule was excited with circularly polarized light and the emitted photons in the s and p channels were measured until the sample bleached (the range of bleaching times was several seconds to 10’s of seconds). Thus, this experiment examines the angular averaging of the transition dipole of the Nile Red, which in turn allows the overall rotational diffusion of the complexes to be obtained on the macrotime scale. Apparently, the Nile Red is in the apolar binding site of the calmodulin protein. So, for considerations of overall rotational diffusion, we assume that the Nile Red transition dipole is fixed to the coordinate axes of the protein and that rotational diffusion of the protein is measured in these experiments. This need not be strictly true for our analyses to be applicable. In reality, there may possibly be some small angular motions of the probe relative to the protein. The fluorescence polarization, A, defined as
A)
Is - RIp Is + RIp
was measured from the fluorescence trajectories of single CaM:peptide-Nile Red complexes. The variables Is and Ip are the fluorescence intensities into the s and p channels, respectively, and R is the factor used to balance the two channels, determined by recording isotropic bulk emission. The distribution of A had a mean of 0 and a full width at half-maximum (fwhm) of 0.17, using this approach. The fwhm of the singlemolecule polarization distributions of CaM:peptide complexes is close to that of some other molecular systems where free rotational diffusion of single molecules were inferred.42,43 The single-molecule polarization distribution was also obtained by calculating the integrated signal from each molecule that appeared in fluorescence images in the two polarization channels. In such a protocol, the overall intensities of the s and p signals from each molecule were obtained and calculated to obtain the polarization A as defined above. The equivalent binning time for each molecule is on the order of 10’s of milliseconds. This method provides a high throughput approach, although the photon counts are limited by the image scanning speed. Furthermore the fluorescence of each molecule is not sampled repeatedly during the image scan. The single-molecule polarization distribution of CaM:peptide complexes obtained in this manner under conditions of 5 mM calcium ion and 2 µM calcium ion are shown in Figure 5. When fitted to Gaussians, both distributions have mean of 0 and a fwhm of 0.3. Factors
Individual Calmodulin:Peptide Complexes
Figure 5. Single-molecule fluorescence polarization as defined in the text as the distribution of CaM:peptide-Nile Red complex with 5 mM calcium-ion concentration (A) and 2 µM calcium-ion concentration (B). Solid curve: the Gaussian fit of experimental data.
that can contribute to the single-molecule polarization distribution width are the rotational diffusion constant (Dr), the shot noise, the binning width, and the experimental background noise. Bulk anisotropy measurements in the hydrogel failed to show any decay of the anisotropy on the time scale of the fluorescence lifetime of Nile Red. At the increased sample concentrations (greater than µM) required for bulk experiments in the hydrogel, the protein tends to aggregate, so the fluorescence depolarization results in the ns time range are not considered reliable. To understand how the distribution of single-molecule fluorescence polarization is related to rotational diffusion of the protein, computer simulations incorporating the photon statistics and rotational diffusion were carried out. Two limiting cases were first considered: In the first, all fluorescent molecules were assumed fixed in space. In the second, the molecules could rotate freely with a rotational correlation time much less than the dataacquisition time. In the former situation, the single-molecule polarization in ideal experiments would show a very broad distribution with peaks at A ) -1 and A ) 1 based on the observed number of photon counts. Because of the limited dynamic range in the intensity measurements, the two peaks would actually be shifted in toward A ) 0.32 In the rapid freemotion limit, again assuming the number of counts observed in this work, the single-molecule polarization would show a very narrow peak around A ) 0, with a fwhm ∼ 0.08 determined entirely by shot noise. The polarization at given photon count rates and time-bin sizes was simulated by means of a simplified Monte Carlo method, assuming the transition dipole is rigidly attached to a protein represented as a spherical diffuser. In general, for every time step ∆t, set as 10 µs, the molecule could rotate from an orientation described by colatitude θ0 and azimuth φ0 to a new orientation (θ, φ) with probability density44
P(θ, φ, ∆t|θ0, φ0) )
Ylm(θ0, φ0)Y /lm(θ, φ) exp(-l(l + 1)Dr∆t) ∑ lm
where Ylm(θ, φ) is the spherical harmonic. When the rotational correlation time is significantly larger than the fluorescence lifetime, the molecule will maintain its orientation throughout the absorption and emission process. The probability of photon emission is proportional to the laser intensity, the absorption coefficient, and the angular factor32 η(θ0)sin2θ0sin2θ, where
J. Phys. Chem. B, Vol. 108, No. 40, 2004 15915
Figure 6. Spectral center distribution of CaM:peptide-Nile Red fluorescence with 5 mM [Ca2+]. Blue curve: the width calculated from photon statistics; red solid line: the Gaussian fit.
η(θ0) is a microscope parameter, with θ0 ) θ for this condition. The probability that the photon is detected in the s or p channels is proportional to cos2φ or sin2φ, respectively. New orientation and emission events were selected iteratively using these probabilities, and the photon numbers were accumulated in s and p channels. The distributions of polarization were then obtained by averaging 1000 randomly chosen initial orientations. This procedure permitted us to estimate the single-molecule polarization distributions expected for different values of the rotational diffusion coefficient. For a binning interval and photon count rate chosen to match the present experiments, it was found that when Dr was greater than 104 s-1, the width of the polarization distribution tended to a value of 0.2. When Dr was less than 104 s-1, the polarization distribution was sensitively dependent on Dr and became broader. Comparisons of these simulations with the data provided a lower-limit estimate of the rotational diffusion coefficient (Dr) of about 3 × 103 s-1, corresponding to an upper limit of the rotational correlation time, Dr/6, of 50 µs. This result confirms the high degree of rotational freedom of CaM:peptide complexes within hydrogel cavities. Moreover, as shown in Figure 5a and 5b, the fluorescence polarization distributions of the CaM:peptide complex at high (5 mM) and low (2 µM) calcium-ion concentration show identical center positions and width for the same count rates and binning width. Therefore, though the complex is possibly undergoing some type of surface hopping (mean residence time < 50 µs) that causes the overall rotational dynamics to have a different character from that in free solution, and though it may have a less-compact structure at the lower calcium concentration, the calcium-ion concentration variations in the current range have no significant effect on the overall motion on the experimental time scale (i.e., microseconds to seconds). If the short-time anisotropy results suggesting there is no motion on the time scale of a few nanoseconds were valid, the surface residence time would be 50 µs-10 ns. Spectra and Lifetime Distributions of the Nile Red Tag in CaM:Peptide at High Ca2+ Concentration. The distribution of peak positions of fluorescence from single CaM:peptideNile Red complexes is shown in Figure 6. The spectral data for each individual molecule were collected, and were averaged over a period of seconds to 10’s of seconds. The histogram represents the number of single molecules having particular values of this average peak frequency. The broadening of this distribution due to photon statistics is indicated by the blue line in Figure 6. This noise contribution was calculated by computer simulations based on a Poisson process. In such a simulation, the expected number of photons emitted into each spectral
15916 J. Phys. Chem. B, Vol. 108, No. 40, 2004
Tang et al.
Figure 7. Fluorescence lifetime distribution of CaM:peptide-Nile Red fluorescence with 5 mM [Ca2+]. Blue curve: the width calculated from photon statistics; red solid curve: the Gaussian fit.
channel, corresponding to the spectral resolution, was calculated from a spectral shape (assumed to be Gaussian) that was the same for each molecule. The total number of photons emitted by each molecule was used as the means to generate spectra assuming a Poisson distribution. About 1000 simulations of spectra and their corresponding peaks were conducted to obtain the width of the distribution of peaks expected for Poisson statistics. The experimental distribution of fluorescence spectral peaks was fitted by a Gaussian profile having a fwhm of 1.0 × 103 cm-1. It is apparent that the experimental distribution is much broader than the statistical noise. Clearly, the environment that is responsible for the spectral shifts is averaging at a rate that is comparable to or slower than the seconds-to-10’s-ofseconds time scale over which the data were collected. More rapid fluctuations of the spectral shifts would narrow the width of the distribution, which would ultimately become determined by photon-counting statistics. The conclusion of very slow dynamics, longer than seconds, is consistent with the expectation that the dissociation/reassociation of single peptides would be slow on the time scale of the measurements. Thus, the breadth of the distribution must be interpreted in terms of conformational heterogeneity on the single-molecule level, which is in slow exchange on the second time scale. A similar conclusion is obtained from an analysis of the distribution of fluorescence lifetimes of single CaM:peptideNile Red complexes, under the condition of 5 mM Ca2+, which is shown in Figure 7. By Gaussian fitting, the fwhm of the lifetime distribution is 0.5 ns. The blue line indicates the broadening due to photon statistics obtained by computer simulations. The strong fluorescence and the extended lifetime of the Nile Red indicate that the tag is partially buried in the apolar pocket of CaM. Since strong Nile Red emission is observed from the CaM:peptide complex, the Nile Red tag must be assumed to be buried within the protein and protected from access to liquid water, which completely quenches the emission of Nile Red to below the current detection limit. The peak of the wavelength distribution is at the same wavelength as the emission maximum in bulk methanol solution, again suggesting a polarity similar to that of methanol. Thus, the probe resides in an environment with water molecules and/or other polar groups that have partial accessibility and undergo restricted reorientation when compared to bulk water. These conclusions are also consistent with the calculations of Carey et al.45 on the hydration structure of the R-chymotrypsin substrate-binding pocket. These calculations showed that the hydrogen bonding strength in the binding pocket decreases by about 50% in free energy, which corresponds to 50% of the hydrogen-bonding power scale ΣR2H. So, the
Figure 8. Fluorescence lifetime distribution of CaM:peptide-Nile Red complexes at different calcium levels. A-F correspond to 0.6 µM, 2 µM, 5µM, 10 µM, 39 µM, and 5 mM [Ca2+], respectively.
hydrogen-bonding strength should be close to a methanol environment (in bulk water, ΣR2H ) 0.82, in methanol, ΣR2H ) 0.43),36 consistent with our single-molecule experimental results. Moreover, using the same peptide as in the present work, Changenet-Barret et al.10 suggested that the response of a probe coumarin dye to fluctuations in the interior of the CaM:peptide could be attributed to confined water molecules. In experiments that confine Nile Red molecules in reverse micelles in which there are water molecules, Datta et al.46 have reported large increases in the fluorescence lifetimes of Nile Red. The measurement time for the spectrum and lifetime of each single molecule in the present case is several seconds to 10’s of seconds. Therefore, the distributions of the spectra and lifetimes represent various molecular conformations that interchange more slowly than this time scale. Dependency of Single-Molecule Fluorescence Lifetimes on Ca2+ Calcium Concentration. The lifetime distributions of single CaM:peptide complexes at various Ca2+ concentrations are shown in Figure 8. These data show clearly that the distributions become wider around the midpoint of the Ca2+ titration curve (fwhm ) 1.7 × 108 s-1), compared with the condition of 5 mM calcium-ion concentration (fwhm ) 0.6 × 108 s-1). Adding Ca2+ also decreases the mean value of the fluorescence decay rate (At 5 mM [Ca2+], kmean ) 3.5 × 108 s-1, while at 2 µM [Ca2+], kmean ) 5.0 × 108 s-1). As discussed above, the fluorescence decay rate is considered to be related to the interaction of peptide-Nile Red with CaM and the protein pocket conformation. At high calcium concentration, the peptide binds tightly to CaM, with the aromatic group of Nile Red anchored into the carboxyl-terminal half of the protein. At intermediate calcium concentrations, the binding will be weaker, increasing the fraction of free unbound Nile Red-peptides. Because of their low quantum yield, the unbound peptides would not be visible in these experiments. A second consequence of the intermediate calcium concentration is increased heterogeneity in terms of the number of calcium ions bound, and, hence, differences in the degree of conformational heterogeneity. The structural variation can readily change the microenvironment of the embedded Nile Red dye. The increase of the fluorescence decay rate is very likely caused by the anchored Nile Red chromophore sensing a more-polar local environment due to the expansion of the binding region. Figure 9 illustrates the relation between the fluorescence decay rate distribution and the hydrogen bond strength of the local environment as indicated in the literature,23 in which the linear relationship between the
Individual Calmodulin:Peptide Complexes
Figure 9. Single-molecule fluorescence decay rate distributions at 5 mM (blue) and 2µM [Ca2+] (red), and their relation to hydrogen bond strength (ΣR2H) and local polarity (ET(30)). The axis for the histograms is not presented due to a space limit. The histograms are identical to the corresponding ones in Figure 8. Please see the text and the cited reference for definitions of these parameters.
hydrogen bonding strength (defined as a function of the equilibrium constants of reactions against a reference series of bases) and the fluorescence decay time of Nile Red was reported. The wider distribution of fluorescent decay rates of Nile Red at intermediate versus high calcium concentration is consistent with the expectation that the ensemble of bound conformers would be more heterogeneous near the midpoint of the calciumbinding isotherm. Furthermore, the shift of the distribution to greater decay rates with decreasing calcium-ion concentration is consistent with a less-complete formation of the hydrophobic binding site on CaM, and, hence, greater exposure of the probe to hydrogen-bonded group and perhaps to some water molecules. Thus, the heterogeneity observed by the single-molecule fluorescence lifetime distribution reveals how the energy landscape47 of the CaM:peptide complex varies in response to the binding of calcium ions. As discussed in the previous section, the wide distribution of single-molecule fluorescence lifetimes indicates that the conformational interchange rate is slower than the measurement time, which is in the range of several seconds. The shapes of these distributions, which have no multimodal character, do not manifest the presence of a small number of states, each having characteristic spectral properties. Thus, the states corresponding to different numbers of bound calcium ions are not distinguished from one another by means of fluorescence lifetime or spectral position. Rather, the heterogeneity is dominated by slowly interchanging conformations within these equilibrium states having bound peptide. Dynamics From PAT Trajectories. The PAT trajectories from ∼50 individual CaM:peptide complexes at 5 mM and 2 µM calcium ion were examined to obtain information concerning molecular motions on the 10 µs-100-ms time scale. The photon-by-photon autocorrelation functions, C2(t), of these two sets of PAT trajectories are shown in Figure 10. These correlation curves describe the evolution of the fluorescence lifetime fluctuations. The difference between the correlation decays at high and midpoint [Ca2+] is significant. The correlation corresponding to high [Ca2+] is flat within the noise level, while that at the midpoint of 2 µM [Ca2+] shows a decay on the time scale of several milliseconds that cannot be fitted to a single exponential. A two-component exponential fit gives τ1 ) 3 ms and τ2 ) 100 ms, and a stretched exponential fit to exp[-(t/ τ)γ] gives τ ) 1.5 ms and γ ) 0.42. The millisecond timescale dynamics from the PAT trajectories of the CaM:peptide complex at 2 µM [Ca2+] are quite different from the dynamics
J. Phys. Chem. B, Vol. 108, No. 40, 2004 15917
Figure 10. PAT correlation analysis of CaM:peptide-Nile Red with 2 µM [Ca2+] (blue cross) and 5 mM [Ca2+] (red cross). Blue line: the stretched-exponential fitting.
that are evident from the lifetime and spectral distributions that must be very slow compared with these measurement times of 1 s to 10’s of seconds. Both results can be understood if the rate of association and dissociation of the peptide from CaM is very slow on the second time scale, but other processes that change the solvent accessibility occur on a more-rapid time scale. Previous kinetic studies of CaM:peptide complex formation and dissociation have been performed by NMR, fluorescence spectroscopy, and other techniques. Two major processes have been investigated: the signal change during the dissociation of calcium ions from CaM34,48,49 and the signal change during the association of CaM and peptide. The second process is triggered either by fastmixing calcium-ion-loaded CaM and peptide50 or by mixing apoCaM:peptide solution and calcium-ion solution.51 These ensemble studies have revealed key features of CaM:peptide complex kinetics; several kinetic models have been proposed based on the typical characteristics found in these experiments. Generally, these models describe the cooperativity of the CaM binding of calcium and peptide by means of discrete state schemes that do not incorporate the diversity of each kinetic component. According to Brown et al.,34 the dissociation rate of calcium ions from the N-terminal of the CaM is 8 s-1, which is much faster than dissociation from the C-terminal. The association rate of calcium ions to the N-terminal of CaM51,52 is in the range of 2 × 108 M-1 s-1. So, at [Ca 2+] ) 2 µM the rebinding rate of calcium ions to the N-terminal of CaM is about 400 s-1. Thus, it is likely that the binding-rebinding of calcium ions contributes to the ms processes observed from our correlation analysis. Furthermore, these processes occur without bindingrebinding of the peptide. These two findings are consistent with the recent work of Wand et al.,53 who found that conformational transitions of bound peptides can occur without full dissociation of the peptide. The present studies show the potential advantages of singlemolecule PAT measurements. There is no dead time limit in these experiments, as occurs in conventional stop-flow or continuous-flow methods. At a fluorescence emission rate of several thousand counts per second, the single-molecule PAT trajectory is an improvement over single-molecule fluorescent intensity recording. This is because the information obtained from each photon is optimized.
15918 J. Phys. Chem. B, Vol. 108, No. 40, 2004 Conclusion Fluorescence spectra and fluorescence lifetimes from CaM:peptide-Nile Red complexes were measured at the singlemolecule level and in bulk samples. The bulk measurements of fluorescence spectrum and lifetime indicate an obvious conformational change based on the significant change of the probe fluorescence as the calcium-ion concentration is changed, because the structural changes of CaM:peptide can alter the local polarity of the microenvironment of probe dye. The probe fluorescence is almost totally quenched by bulk water when the labeled peptide is in the aqueous phase, out of the CaM binding pocket. The fluorescence lifetime of the Nile Red probe is found to be a useful measure of local hydrogen-bond strength and local polarity. The same sensitivity is not apparent in the spectral shifts. The single-molecule experiments yielded the distribution of single-molecule fluorescence spectral peak positions and lifetimes of CaM:peptide-Nile Red complexes. The results expose significant heterogeneity of CaM:peptide complexes that persists for times in the 1-s-to-10’s-of-seconds regime. The range of protein conformations of the CaM:peptide complex can be assigned to a range of local hydrogen-bonding strength or polarity by comparisons with other experiments on the probe Nile Red. In addition, the single-molecule dynamics of CaM:peptide complexes have been studied by means of fluorescence-lifetime imaging and photon-arrival time trajectories. From fluorescence lifetime imaging, the lifetime distribution of CaM:peptide-Nile Red complexes at different Ca2+ ion concentrations manifested the heterogeneity of the CaM:peptide conformation at different calcium concentrations. At Ca2+ concentrations near the middle point of the calcium titration curve, the variance of lifetime distribution of the Nile Red probes in CaM binding pocket is four times larger than that at high calcium concentration. This result is attributed to changes in the free-energy surface with calcium-ion concentration. At intermediate calcium-ion concentrations, a heterogeneous ensemble of CaM:peptide complexes with fewer than four Ca2+ per CaM is formed. The heterogeneity of the ensemble, as well as the rates of interconversion, can be determined using single-molecule studies. The PAT trajectories indicate the dynamic difference between the peptide dissociation from the CaM:peptide complex at high and low calcium levels. At [Ca 2+] ) 2 µM, the midpoint of the titration, the correlation time of the fluorescence lifetime fluctuation is several milliseconds, while at 5 mM [Ca 2+] there is no significant decay of the correlations from the fluorescence lifetime fluctuation on time scales greater than 0.1 ms or less than ca. 0.1 s. A dynamic disorder, implying conformational transitions of bound peptides within the CaM:peptide-Nile Red complexes, is disclosed by the nonexponential character of the fluorescence-lifetime correlation decay. Because the bright fluorescence only appears when Nile Red is protected from liquid water, the current methods should have broad applicability for studying protein folding as well as ligand protein interactions. Acknowledgment. This research was supported by NIH grant PO1GM048130 to W.F.D. and R.M.H. using instrumentation developed under Resource Grant RR001348 to R.M.H. References and Notes (1) Eizenberg, N.; Klafter, J. Chem. Phys. Lett. 1995, 243, 9. (2) Jia, Y.; Sytnik, A.; Li, L.; Vladimirov, S.; Cooperman, B. S.; Hochistrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7932. (3) Lu, H. P.; Xun, L.; Xie, X. S. Science 1998, 282, 1877.
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