Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

Aalim M. Weljie and Hans J. Vogel1

Department of Biological Sciences, University of Calgary,2500 University Drive NW, Calgary, T2N 1N4, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca2+-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM–MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM–CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.

Keywords: calmodulin/ethionine/methionine/norleucine/selenomethionine/tryptophan fluorescence


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The understanding of calcium-mediated cell signaling has advanced rapidly over the past decade, and it has become clear that calmodulin (CaM) plays a complex role in these processes (for reviews see Means et al., 1991; Vogel, 1994; Crivici and Ikura, 1995; James et al., 1995; Vogel and Zhang, 1995; Ikura, 1996). CaM is a pervasive acidic 16.7 kDa bilobal protein which binds two calcium ions in each half of the molecule. The two lobes are connected by a central linker region which is helical in the crystal structure of Ca2+-saturated CaM (Babu et al., 1988Go), but has been shown to be flexible in solution (Barabato et al., 1992Go; van der Spoel et al., 1996Go). This protein has been implicated in binding to over 40 different substrates, and activation of numerous target enzymes (Vogel, 1994Go) in response to transient increases in cytosolic Ca2+ concentration. Of particular interest for the CaM system is the mode of recognition for such a multitude of targets, given that the primary sequence for most CaM-binding regions identified in target proteins lack significant amino acid sequence homology.

In general, calcium binding is required in the CaM protein prior to substrate binding. Structures of the apo- and Ca2+ forms of CaM show that there are two hydrophobic pockets which form upon binding calcium (Babu et al., 1988Go; Kuboniwa et al., 1995Go; Zhang et al., 1995Go). To date, there has not been an X-ray or NMR structure of an intact CaM-target protein complex reported in the literature. Our understanding of the CaM-target binding mode is limited to complexes of CaM with synthetic peptides encompassing the CaM-binding regions from myosin light chain kinases (MLCKs) and CaM-dependent protein kinase II (CaMKII) (Ikura et al., 1992Go; Meador et al. 1992Go, 1993Go; Clore et al., 1993Go). In the presence of a target peptide, a complex is formed in which the target peptide adopts an {alpha}-helical conformation (Seeholzer, 1986; Roth et al., 1991Go; Clore et al., 1993Go; Zhang and Vogel, 1994aGo), and the two lobes of CaM collapse around the target in a cis conformation, perturbing the central linker region.

Studies have shown that many CaM binding target regions have potential to form basic amphiphilic {alpha}-helices (O'Neil and DeGrado, 1990Go). The CaM–MLCK and CaM–CaMKII structures support the conclusion that the mode of interaction between CaM and its targets is built upon a combination of hydrophobic and electrostatic interactions. Of particular interest in the understanding of how CaM interacts with such an abundance of substrates is the role of methionine residues (O'Neil and DeGrado, 1990Go; Vogel, 1994Go; Crivici and Ikura, 1995Go; Vogel and Zhang, 1995Go). In the Ca2+–CaM–MLCK complexes, the Met side chains contribute roughly 50% of the binding surface area to the target peptides (Ikura et al., 1992Go; Meador et al., 1992Go). Met is thought to play a particularly important role in multi-target proteins such as CaM and the signal-receptor particle (SRP54). The lack of steric hindrance and the presence of the polarizable sulfur atom in the side chain allows for a greater range of side chain conformations, providing an easily polarized `sticky' surface based on van der Waals' interactions (Gellman, 1991Go; Zhang and Vogel, 1994aGo; Yuan et al., 1998Go, 1999aGo).

Another important element in binding of specific substrates to CaM is an aromatic residue, frequently tryptophan, in the CaM-binding region of the target. It has been thought that the nonpolar aromatic residue acts as an anchor into one of the hydrophobic patches from CaM (O'Neil and DeGrado, 1990Go; Graether et al., 1997Go). Figure 1Go shows the Trp from smooth muscle MLCK (smMLCK) interacting with the Met residues from the C-terminal lobe of CaM. It is evident that the interactions between the Met residues and the Trp are substantial. Fluorescence spectroscopy has previously been shown to be a sensitive tool for monitoring changes in the local environment of tryptophan residues in CaM-binding peptides (O'Neil et al., 1987Go). We have monitored the interaction between synthetic skeletal muscle MLCK (skMLCK) and CaMKI CaM-binding peptides with wild-type CaM (WT-CaM), selenomethionine-CaM (Se-CaM) and a quadruple methionine to leucine C-terminal mutant of CaM (CT-CaM)(Yuan et al., 1998Go). The systems under study are especially well suited to tryptophan fluorescence studies as CaM lacks this residue, and there is a unique tryptophan in many of the CaM-binding peptides which can be exclusively monitored. The tryptophan fluorescence emission maximum exhibits a marked blue shift (~ 20 nm) upon CaM binding, as compared with the maximum emission wavelength for the free peptide. Upon binding to CT-CaM, there is a marked increase in the fluorescence intensity, presumably due to the constraints imposed by the more rigid leucine side chains. There is an opposite effect when selenomethionine is incorporated instead of Met, which is probably due to the quenching ability of the large and polarizable selenium atom (Yuan et al., 1998Go).



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1. Space-filling representation of the interaction between tryptophan from MLCK and the C-terminal Met residues from CaM. The {alpha}-helical MLCK peptide backbone is shown as a ribbon diagram in addition to the Trp side chain. The Trp is packed into the C-terminal lobe of CaM, and sequestered by Met109 and 124 (above the plane of the ring) and Met144 and 145. Each of these Met residue side chains are labeled near the key sulfur atom. Coordinates were taken from the Brookhaven Protein Databank from the crystal structure of Meador et al. (1992), and the figure was prepared with MOLMOL (Koradi et al., 1996Go).

 
This paper extends our previous work (Yuan et al., 1998Go) to include several systems for which the crystal structures of CaM-target peptide complexes are not known. This includes the interaction of CaM with synthetic peptides encompassing the CaM-binding regions of caldesmon site A and B (CaD A and CaD B, respectively), cyclic nucleotide phosphodiesterase (PDE) and melittin. CaD is an actin binding protein which has been implicated in the regulation of smooth muscle contraction (Huber, 1997Go). Cyclic nucleotide PDE is implicated in cyclic adenine monophosphate and cyclic guanosine monophosphate hydrolysis (Charbonneau et al., 1991Go). Melittin is a CaM antagonist derived from honey bee venom (Comte et al., 1983Go). We have also extended the scope of this study to include CaM containing the unnatural amino acids ethionine and norleucine, which were biosynthetically substituted in place of methionine (Figure 2Go). These proteins fold properly and give rise to enzymatic activation (Yuan and Vogel, 1999Go).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Structures of the methionine analogues used in this study which were biosynthetically incorporated as described in the Materials and methods section. The key atomic changes are highlighted in bold type. Typical bond lengths and angles are as follows: C–C, 1.50 Å, CCC 114°; C–S, 1.74 Å, CSC 109°; C–Se, 1.93 Å, CSeC 106° (Bowen, 1958). The lengthening and flattening of the C–S–C and C–Se–C bonds leads to significantly different chemical and physical properties, as evidenced by the enthalpic barriers to rotation which change according the order: CC–CC, 0.81 kcal/mol; CS–CC 0.22 kcal/mol; CSe–CC, 0.14 kcal/mol (Ohno et al., 1980Go). The lower CS–CC rotation energy is hypothesized to increase the malleability of Met, as the conformational flexibility increases as the energetic barrier decreases (Gellman, 1991Go).

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Proteins

Mammalian WT-CaM and CT-CaM were purified from Escherichia coli using a double hydrophobic chromatography method described elsewhere (Vogel et al., 1983Go; Waltersson et al., 1993Go; Zhang and Vogel, 1993Go). The proteins were further purified by application to a Sephadex G-100 column (2 cmx100 cm), and eluted with NH4CO3 pH 8.0, and subsequently lyophilized for desalting. Eth-CaM and Nleu-CaM were expressed and purified in the same manner as previously described for the Se-CaM protein (Zhang and Vogel, 1994bGo). Incorporation of Se-Met was determined by NMR spectroscopy and amino acid analysis. Incorporation of other unnatural amino acids was assessed by amino acid analysis and mass spectrometry. Purity was confirmed by SDS–PAGE (not shown). The concentration of all protein samples was determined by UV spectroscopy, using {varepsilon}1%276 for CaM of 1.8.

The synthetic peptides used in this study were based on the following known CaM-binding regions: skeletal muscle MLCK 577–598, KRRWKKNFIAVSAANRFKKISS (Blumenthal et al., 1985Go); cyclic nucleotide phosphodiesterase, Ac-QTEKMWQRLKGILRCLVKQL-NH2 (Charbonneau et al., 1991Go); melittin, GIGAVLKVLTTGLPALISWIKRKRQQ (Compte et al., 1983); caldesmon A site, GVRNIKS- MWEKGNVFSS (Zhan et al., 1991Go); caldesmon B site, NKETAGLKVGVSSRINEWLTKT (Marston et al., 1994Go). Synthesis was performed at the Core Facility for Protein/DNA Chemistry, Queen's University, Canada. Purity was assessed to be >95% by HPLC and amino acid analysis. Peptide concentrations were determined based on the UV absorption of the sole tryptophan residue, based on {varepsilon}280 = 5500 M–1 cm–1.

Fluorescence spectroscopy

Fluorescence data were collected on a Hitachi F-2000 spectrofluorimeter, with emission and excitation bandpasses set to 10 nm. Tryptophan excitation emission was set to 295 nm to minimize interference from CaM tyrosine fluorescence, and emission spectra were recorded from 300 to 400 nm. All samples contained 10 mM Tris–HCl pH 7.4 and 100 mM KCl. Calmodulin proteins and binding peptides were added from 0.50 and 1 mM stocks to final concentrations of 12 and 10 µM respectively. CaCl2 was added to samples to a final concentration of 1 mM. The reversibility of CaM–target fluorescence was determined by the addition of EDTA to a final concentration of 5 mM. All final spectra recorded were difference spectra taking into account native CaM fluorescence and buffer effects. Under these conditions, the fluorescence observed can be attributed to the sole tryptophan from the CaM-binding peptides. Quenching data were collected on the (Ca2+)4 –CaM–target complexes and the free target peptides. Aliquots (20 µl) of a 5 M KI stock were added to 1 ml samples to a maximum concentration of 2.5 M. The recorded emission intensities were corrected for dilution effects.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to probe the interaction of CaM with several peptides encompassing the CaM-binding domains of CaM–target proteins, we have developed a strategy to incorporate the unnatural amino acids selenomethionine, norleucine and ethionine in place of Met residues. We have also used a quadruple mutant protein with four C-terminal Met residues converted to Leu.

Protein purification

Purification of all CaM proteins (WT-CaM, CT-CaM, Se-CaM, Nleu-CaM and Eth-CaM) was successful based on the method of calcium-dependent double hydrophobic chromatography. Amino acid analysis data indicate that biosynthetic incorporation of ethionine and norleucine in place of methionine was ~ 85% (data not shown), similar to that determined previously for selenomethionine incorporation (Zhang and Vogel, 1994aGo; Yuan et al., 1998Go). All methionine analogue proteins displayed Ca2+-dependent SDS–PAGE bandshifts (Klee et al., 1979Go) similarly to WT-CaM (data not shown).

Fluorescence spectroscopy

The steady state tryptophan fluorescence of the five CaM variants with five CaM-target peptides was monitored, based on the Trp residue from the CaM-binding sequences. In all cases, there was a blue-shift in the observed emission maximum upon CaM–target complex formation as compared with the maximum fluorescence emission peak from the free peptides consistent with the placement of the Trp residue in a hydrophobic environment. The extent of the blue-shift however was strongly dependent on both the peptide and the form of CaM under study (Table IGo).


View this table:
[in this window]
[in a new window]
 
Table I (a) Blue-shift observed in tryptophan fluorescence by various CaM analogue protein–target complexes (nm); (b) Tryptophan fluorescence maxima observed for free peptides and CaM–target complexes (nm)
 
MLCK peptide fluorescence

We have previously reported that when WT-CaM forms a complex with a synthetic peptide encompassing the CaM-binding domain from MLCK there is a substantial blue-shift of 22.0 nm in the tryptophan fluorescence emission maximum (Yuan et al., 1998Go). This is consistent with the observed shift of 21.5 nm in this experiment (Figure 3Go, Table IGo), from 353.5 nm for the free MLCK peptide to 335.0 for the WT–CaM–MLCK complex. As observed in our earlier studies (Yuan et al., 1998Go), Se-Met was also found to be an efficient quenching agent of Trp fluorescence, while maintaining a similar blue-shift in the emission maximum (22.5 nm). The CT–CaM–MLCK complex exhibits a comparable shift in Trp fluorescence emission maximum (20.5 nm) while showing greatly increased intensity. These observations are consistent with the crystal and NMR structures of CaM–MLCK complexes in which the Trp residue of the MLCK peptide is inserted in the hydrophobic cleft of the C-terminal end of calcium–CaM, where it is surrounded by Met residues (Figure 1Go; Meador et al., 1992; Ikura et al., 1992). The complexes with the MLCK peptide of Nleu-CaM and Eth-CaM also give rise to significant blue-shifts of 23.0 and 15.0 nm respectively. The emission maximum blue-shift with Eth-CaM, while significant, was substantially less ( ~ 7 nm) than the blue shift observed with the other calmodulin proteins. In order to determine if the observed shift was due to a different binding mode, dynamic bi-molecular quenching studies were conducted with MLCK and all five CaM proteins (Figure 4Go). The Stern–Volmer quenching constants derived from these studies clearly indicate that the mode of binding for Eth-CaM is essentially the same as that of WT-CaM and the other CaM proteins used in this study. These data suggest that the solvent accessibility of the tryptophan residue in the MLCK peptide is essentially the same in all complexes (Lakowicz, 1983Go), and that observed differences in blue-shifts are not due to solvent effects.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. Emission spectrum from the steady state tryptophan fluorescence of the MLCK peptide and Ca2+–CaM–MLCK complexes. The MLCK peptide alone ({blacksquare}), WT-CaM–MLCK ({blacktriangledown}), CT-CaM–MLCK (•), Se-CaM-MLCK ({square}), Nleu-CaM–MLCK ({blacktriangleup}) and Eth-CaM–MLCK ({blacksquare}). Protein and peptide concentrations were 12 and 10 µM respectively.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Stern–Volmer plots of the KI bi-molecular tryptophan fluorescence quenching of MLCK peptide and Ca2+–CaM–MLCK complexes. Stern–Volmer quenching constants (M–1) normalized to free peptide fluorescence were 1.00, MLCK peptide alone ({blacksquare}); 0.08, WT-CaM–MLCK ({blacktriangledown}); 0.04, CT-CaM–MLCK (•); 0.13, Se-CaM–MLCK ({square}); 0.04, Nleu-CaM–MLCK ({blacktriangleup}) and 0.09, Eth-CaM–MLCK ({blacksquare}). Protein and peptide concentrations were 12 and 10 µM respectively. Fo is the fluorescence intensity in the absence of KI, and F is the dilution-corrected intensity observed at each titration point.

 
Melittin peptide fluorescence

In terms of the observed blue-shift, the results of the interaction of melittin with the various CaM proteins were similar to those with MLCK, with the exception of the Se-CaM (Figure 5Go). In this case, the observed blue-shift (9.5 nm) was almost half those of the WT-CaM and CT-CaM (18.0 and 18.5 nm respectively), and less than that of Nleu-CaM and Eth-CaM (23.0 and 12.5 nm).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Emission spectrum from the steady state tryptophan fluorescence of the melittin peptide and Ca2+-CaM–melittin complexes. The melittin peptide alone ({blacksquare}), WT-CaM–melittin ({blacktriangledown}), CT-CaM–melittin (•), Se-CaM–melittin ({square}), Nleu-CaM–melittin ({blacktriangleup}), and Eth-CaM–melittin ({blacksquare}). Protein and peptide concentrations were 12 and 10 µM respectively.

 
Earlier studies of the CaM–melittin complex (Scaloni et al., 1998Go; Seeholzer et al., 1987Go) have shown that the binding mode of melittin to CaM occurs in a parallel manner, meaning that the N- and C-terminal regions of the peptide associate with the amino and carboxy domains of the protein respectively. Thus the Trp residue of melittin is situated near the C-terminus of the peptide (Table IIGo), it is somewhat surprising that there is no significant intensity increase (4%) in the tryptophan fluorescence of the CT–CaM–melittin complex. This result highlights the complexity, and the lack of a detailed understanding, in interpreting fluorescence intensity data.


View this table:
[in this window]
[in a new window]
 
Table II. Overview of CaM-binding peptide sequences and binding orientation
 
Cyclic nucleotide phosphodiesterase peptide fluorescence

The tryptophan fluorescence emission spectrum of the uncomplexed PDE peptide is unique, as the maximum wavelength was already significantly blue-shifted compared with the other peptides under study (Figure 6Go; free PDE, 344 nm, cf. 354 for free MLCK, melittin and CaD A, 355 for CaD B). The CaM–PDE complexes demonstrated Trp emission maxima which were similar to those observed from the CaM–MLCK complexes. For example, the emission maximum of the WT–CaM–PDE complex was 332 nm (Table IbGo), compared to the WT–CaM–MLCK complex which was 333 nm. The Se–CaM–PDE complex was notably deviant from this trend, exhibiting a significantly smaller blue shift (337 nm, cf. 332 nm for the Se-CaM-MLCK complex). The CaM-binding sequence from PDE has some inherent property which seems to prevent stabilization of the excited state to the same extent as the other peptides in the free form. Examination of the primary sequence does not provide any immediate indication as to why this may be, although this is the only peptide with a Cys residue present.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6. Emission spectrum from the steady state tryptophan fluorescence of the PDE peptide and Ca2+-CaM–PDE complexes. The PDE peptide alone ({blacksquare}), WT-CaM–PDE ({blacktriangledown}), CT-CaM–PDE (•), Se-CaM–PDE ({square}), Nleu-CaM–PDE ({blacktriangleup}) and Eth-CaM–PDE ({blacksquare}). Protein and peptide concentrations were 12 and 10 µM respectively.

 
Caldesmon site A and B peptide fluorescence

The relative shift in the emission wavelength maxima for all five CaM–CaD A complexes was typical of the other peptides examined (Figure 7Go). The shift of the complexes of CaD A with WT-CaM, CT-CaM and Eth-CaM were within 1.5 nm. The Se-CaM shift was slightly lower (by 3.5 nm). Also consistent with previously discussed CaM–targets in this study, the Eth-CaM complex shows a relative blue-shift in the emission wavelength maximum which is essentially half that of the WT–CaM–CaD A complex (9.5 nm cf. 20.0 nm).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7. Emission spectrum from the steady state tryptophan fluorescence of the CaD A peptide and Ca2+-CaM–CaD A complexes. The CaD A peptide alone ({blacksquare}), WT-CaM–CaD A ({blacktriangledown}), CT-CaM–CaD A (•), Se-CaM–CaD A ({square}), Nleu-CaM–CaD A ({blacktriangleup}) and Eth-CaM–CaD A ({blacksquare}). Protein and peptides concentrations were 12 and 10 µM respectively.

 
The Trp fluorescence emission spectra of the CaM–CaD B complexes were anomalous in comparison with the four other peptides under study (Figure 8Go). The blue-shifts for the complexes ranged from 2.0 nm with Se–CaM–CaD B to 18.0 nm with CT–CaM–CaD B. Particularly noteworthy was the Se–CaM–CaD B shift, which at 2.0 nm was indicative of a minor tryptophan environmental change. Supporting this notion was the fact that the intensity of fluorescence for this complex was essentially unquenched in comparison to the free peptide, suggesting that the selenomethionine residues were not within quenching distance of the peptide tryptophan. In order to determine if the CaM–CaD B complex was binding in an analogous manner to the CaM–MLCK complexes, further Stern–Volmer quenching studies were performed as shown in Figure 9Go. It was obvious that the Trp from the CaD B peptide is not sequestered by the CaM proteins, and is accessible to the quenching agent (Lakowicz, 1983Go). An interesting result of this experiment is the indication of dynamic quenching, based on positive curvature in the slopes of the quenching curves of the complexes. In the case of static quenching, (such as a free indole moiety in a polar solution), the Stern–Volmer curve is expected to be linear, which is consistent with our results from CaM–MLCK quenching experiments (Figure 4Go). However, the presence of the curvature in the quenching curve as with the CaM–CaD B complexes indicates an association between some entity (either protein or peptide) and the quenching agent, in this case iodide ions. These quenching results suggest that the tryptophan from the CaD B site is not well protected, which is also consistent with the data from fluorescence emission maximum wavelength blue-shifts.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8. Emission spectrum from the steady state tryptophan fluorescence of the CaD B peptide and Ca2+-CaM–CaD B complexes. The CaD B peptide alone ({blacksquare}), WT-CaM–CaD B ({blacktriangledown}), CT-CaM–CaD B (•), Se-CaM–CaD B ({square}), Nleu-CaM–CaD B ({blacktriangleup}) and Eth-CaM–CaD B ({blacksquare}). Protein and peptides concentrations were 12 and 10 µM respectively.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 9. Stern–Volmer plots of the KI bi-molecular tryptophan fluorescence quenching of CaD B peptide and Ca2+-CaM–CaD B complexes. Stern–Volmer quenching constants (M–1) normalized to free peptide fluorescence were 1.00, CaD B peptide alone ({blacksquare}); 0.74, WT-CaM–CaD B ({blacktriangledown}), 0.50, CT-CaM–CaD B (•); 0.67, Se-CaM–CaD B ({square}), 0.61, Nleu-CaM–CaD B ({blacktriangleup}) and 0.74, Eth-CaM–CaD B ({blacksquare}). Protein and peptides concentrations were 12 and 10 µM respectively. Fo is the fluorescence intensity in the absence of KI, and F is the dilution-corrected intensity observed at each titration point. The effect of curvature was neglected in the calculation of the quenching constants.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has long been known that fluorescence spectroscopy can be a sensitive tool in probing the interaction of calmodulin with target peptides containing Trp residues (O'Neil et al., 1987Go). We have used this tool to monitor the interaction of CaM with several peptides which encompass the CaM-binding domain from known CaM–target proteins. Furthermore, using biosynthetic methods to incorporate unnatural amino acids, we have probed the effect of substituting Met residues with various analogues on this CaM–target interaction.

All proteins used in this study were successfully purified via double phenyl-Sepharose hydrophobic chromatography, in a manner analogous to that used for the purification of WT-CaM. To a first approximation, this suggests that the hydrophobic clefts which CaM exposes at either lobe of the protein behave in a similar calcium-dependent manner. We have previously shown for Se-CaM that there is a minimal perturbation of the structure and function as compared with WT-CaM by NMR and enzyme activation studies (Zhang and Vogel, 1994bGo; Vogel and Zhang, 1995Go), and likewise for norleucine and ethionine (Yuan and Vogel, 1999Go). Also the quadruple Met to Leu mutant CT-CaM is structurally similar and can activate many target binding proteins (Zhang et al., 1994Go; Edwards et al., 1998Go).

Fluorescence transitions

Recent quantum mechanical calculations of excited state indole systems have characterized the energy transitions which give rise to particular wavelength shifts in fluorescence methods (Callis, 1997Go). Of importance is the ability of the environment surrounding the fluorophore to respond to the excited state dipole induced by excitation. An instantaneous response (relative to the fluorescence lifetime) by the environment, such as in a polar solvent, will stabilize the excited state, and result in fluorescence emission that is red-shifted as compared with a nonpolar environment (Lakowicz,, 1983Go; Callis, 1997Go). In principle this result can be applied to the CaM–target system, where the local environment of a Trp from a CaM-binding peptide will be massively influenced by the nature of the hydrophobic cleft from CaM. Hence the depth of the fluorescence transition, and the resultant blue-shift in Trp fluorescence relative to free peptide, can be interpreted based on the stabilization (or lack thereof) at the level of the excited state. Such electronic dipolar interactions are distinct from bi-molecular quenching, a phenomenon in which the excited state of the fluorophore is relaxed by a non-radiative process (Lakowicz, 1983Go; Chen and Barkley, 1998Go), which affects the quantum yield, hence the intensity, of the observed fluorescence.

Ethionine calmodulin

To interpret the results of this study, it is critical to develop an in-depth understanding of the chemical properties of each Met analogue or substitution which impart potential changes to the hydrophobic binding cleft of CaM, and the subsequent Trp fluorescence. The most obvious steric change comes from the ethionine residue which contains an extra methylene group between the {delta}-sulfur atom and the terminal methyl group (Figure 2Go) thus extending the amino acid side chain by 3 Å (Alix, 1982Go). There are close contacts between the Met residues from CaM and the Trp from MLCK in the crystal structure of the complex (<=7 Å between the center of the Trp ring and the Met sulfur atoms (Yuan et al., 1998; Figure 1Go). The introduction of these side chain extensions may perturb the side chain packing arrangement in the complex, conferring some conformational flexibility to the Trp residues not present with Met side chains. Such conformational flexibility would give rise to a decrease in the extent of fluorescence blue-shift (Lakowicz, 1983Go; Callis, 1997Go), as there would be additional stabilization interactions of the excited state not allowed in the more rigidly packed native structure. The blue-shift observed for the other Eth-CaM complexes is consistent with the data from the MLCK complex, each of them showing significantly smaller blue-shifts than the respective WT-CaM complexes. This provides further evidence that the Met residues are critical in ensuring that the CaM protein interacts specifically with each of its targets, and that the packing in each complex is not completely tolerant to the introduction of additional moieties. In order to further probe these interactions between the ethionine residues and the Trp moiety from the MLCK peptide, selenoethionine was incorporated into CaM. The fluorescence data from this protein (data not shown) show that the Trp fluorescence is quenched. Hence we conclude that the selenium groups of selenoethionine are close enough to the Trp residue such that the packing is not perturbed significantly, supporting the CaM–MLCK quenching results which demonstrated that the Trp from the peptide is not significantly solvent exposed.

Norleucine calmodulin

The results of the Nleu–CaM complexes are interesting as they illustrate the specificity of CaM for its targets, and supports the notion that there are multiple binding modes for CaM–target complexes. The norleucine molecule is only slightly different in terms of size with the substitution of the Met {delta}-sulfur with a methylene group, however norleucine is significantly different from Met due to conformational restrictions in the {chi}3 torsion angle involving the {delta} position of the sidechain (Gellman, 1991Go). The CS–C bond shows little preference for the gauche or anti conformation based on enthalpy in Met; however, in contrast, the CC–C bond of norleucine favours the anti conformation, presumably for steric reasons. Such preference would impart a degree of rigidity to the norleucine, as opposed to the `plasticity' of the Met residue. The data of this study suggest that such a change in steric properties may not allow the binding pocket of CaM to adjust specifically to each target, enhancing some interactions and hindering others. Also, the change from sulfur to methylene will change the polarity of the side chain, presumably increasing the hydrophobicity, although if the packing interactions are disrupted this effect would be expected to be minor. In the case of the Nleu–CaM–MLCK complex it appears as though this conclusion is supported by the blue-shift of Nleu–CaM–MLCK which is 1.5 nm greater than that of WT-CaM (23.0 cf. 21.5 nm); the replacement of the sulfur by a methylene group does not seem to upset the packing interactions, while the blue-shift increase predicts that the excited state of the Trp residue experiences a fairly electronically static environment, presumably imparted by the increased hydrophobicity of the norleucine side chains. The other Nleu–CaM–peptide complexes, with the exception of the CaD B peptide (see below), show opposite relative Trp fluorescence blue-shifts compared with their native equivalents, with the modified proteins causing slightly smaller blue-shifts, the most pronounced of which is the Nleu–CaM–melittin complex. In these cases perhaps the rigidity of the norleucine side chains hinders the hydrophobic binding cleft from CaM in packing to the same extent as in the WT-CaM complexes.

Selenomethionine calmodulin

The structural change induced by the incorporation of selenomethionine in place of Met is presumably minimal, based on our previous work on CaM systems (Zhang and Vogel, 1994aGo ; Yuan et al., 1998Go), and the popularity of selenomethionine as a heavy atom in X-ray crystallographic structural studies (Ogata, 1998Go). With this modification, a large, electron rich and polorizable selenium atom replaces the sulfur in methionine. It is surprising then that there is a large variance in the magnitude of the blue-shifts observed in the Se-CaM–target complexes studied here. As we have previously reported, the complex of Se-CaM–MLCK shows a similar blue-shift compared with the native complex, although the intensity of fluorescence is vastly diminished, presumably due to the bi-molecular quenching of Trp fluorescence by the selenium group (Yuan et al., 1998Go). Substantial quenching of fluorescence was also observed in the Se-CaM complexes with melittin and PDE; however, only marginal quenching was seen with Se-CaM and the CaD A and B peptides. Interpretation of the blue-shifts suggest that in the case of the Se-CaM and melittin, PDE, CaD A and B complexes, the selenium atoms are situated in a manner which would allow for an immediate stabilization of the Trp excited state. This is in contrast to MLCK, where the Se-CaM complex shows a blue-shift very similar to that with WT-CaM. Of the peptides studied, MLCK has the strongest binding constant with CaM (Table IGo), and hence this result may be due to a tight `pinching' of the Trp side chain by the selenomethionine side chains in such a manner that an immediate electronic dipole stabilization is not possible upon excitation of the fluorophore, possibly due to interference from the terminal methyl group in selenomethionine. When compared with the other peptides used in this study (Table IGo), it is evident that the Se-CaM–melittin complex shows the smallest relative blue-shift compared with the WT equivalent, with the exception of Se-CaM–CaD B which does not bind tightly (see CaD discussion later in this section). The lower affinity of CaM for the other peptides used compared with MLCK implies that the selenomethionine and indole groups have more conformational motion within the binding cleft of these CaM–target complexes, leading to increased dipole stabilization by the mobile selenium groups upon fluorescence excitation, and a consequent reduction in the blue-shift as compared with the native complexes. All the same, it is interesting that the blue-shift of the Se-CaM–melittin complex is only about half that of the WT-CaM–melittin complex, given that the binding constant for CaM–melittin is 3 nM (Table IGo), which is much lower than that of PDE and CaD which show such more pronounced blue-shifts. This result further indicates that CaM indeed has distinct recognition abilities for individual peptides, and that the indole moieties from peptide Trp bind in a heterogeneous manner with respect to CaM Met and selenomethionine residues.

Calmodulin mutant protein

The CT-CaM mutant protein has the unique feature of containing a wild-type N-terminal lobe, with all methionines substituted to leucines in the C-terminal lobe. Hence one would expect that the binding and consequential fluorescence would be indicative of the position of the Trp residue from the peptide within the complex. We have shown that there is a substantial increase in fluorescence intensity within the CT-CaM–MLCK complex (Yuan et al., 1998Go), presumably due to the localization of the Trp from MLCK in the C-terminal lobe of CaM, which was also seen in this work. In this complex, as well as all others except CT-CaM–CaD B (see below), the blue-shift was essentially the same as that of the respective WT-CaM–target complexes. For the CT-CaM–MLCK and CT-CaM–PDE complexes, this suggests that the Leu residues provide the same level of dipole stabilization for the excited state of the Trp fluorophore as Met from the wild-type complexes. It was intriguing that there was only nominal intensity changes in the CT-CaM–PDE and melittin complexes, especially in light of the CT-CaM–MLCK complex which shows a major intensity increase compared with the native complex. In all three complexes the Trp residue from the peptide is expected to be in the C-terminal lobe of CaM upon complex formation (Table IIGo). This can not be explained by the affinity of binding between the various CaM targets (Table IIGo), and hence must be related to the differences in the nature of binding within the hydrophobic cleft. Recent work done to examine the factors which affect Trp fluorescence in proteins has shown that there are numerous non-radiative de-excitation pathways from the excited state of the fluorophore which can contribute to intensity changes in fluorescence (Chen and Barkley, 1998Go). Any analysis of the intensity data is hence rendered speculative, unless the existence of each of these pathways is examined individually, which is certainly beyond the scope of the present study. There is however extensive biochemical evidence which shows that the interactions with CaM and PDE or MLCK are very different (Zhang et al., 1994Go; Torok et al., 1998Go; Yuan et al., 1999).

The CT-CaM–CaD B peptide complex is particularly striking as it is the only CT-CaM complex which shows an increased blue-shift for the emission maximum as compared with the WT-CaM complex. This is intriguing given that the binding of the CaD B peptide to CaM is weaker than MLCK by over 103-fold (Table IIGo), and the quenching results indicate that binding to CaM does not protect the Trp residue in the CaD B peptide from the solvent (Figure 9Go). Further examination of the bi-molecular quenching does show however that the solvent accessibility of the Trp residue is less in the CT-CaM–CaD B complex than any other complex, including WT-CaM–CaD B. It has become apparent from recent studies of CaM–CaD interactions that multiple domains of CaD are implicated in binding (Huber et al., 1996Go; Graether et al., 1997Go; Zhou et al., 1997Go). It has been found that the CaM–CaD interaction occurs in an antiparallel manner, with site A (N-terminal) and site B (C-terminal) interacting preferentially with the CaM C- and N-termini respectively (Wang et al., 1997Go; Zhou et al., 1997Go). This multi-domain interaction is unique to caldesmon in terms of CaM-binding targets, and provides some explanation as to the unusual results observed with the two CaD peptides. It has previously been shown that the CaM–CaD A interaction is ~1000-fold weaker than typical CaM–target interactions (Kd in the µM range); in addition, it has been found that the CaD site B peptide binds CaM 3-5-fold more weakly than the site A peptide (Graether et al., 1997Go), and that only the C-terminal half of the CaD B peptide becomes helical upon forming a complex with CaM (Zhou et al., 1997Go). Our fluorescence results support the notion that the binding mode of CaD to CaM is atypical of CaM-binding entities, and that in particular the CaD B site gives markedly distinct fluorescence spectra. The increase in blue-shift observed in the CT-CaM–CaD B complex is also consistent with an interaction that is specific for the C-terminal domain of the CaM protein.

Concluding remarks

Taken as a whole, our results support the conclusion that Met residues provide a particularly malleable surface for target proteins of CaM, and are one of the major contributing factors of the ability of CaM to recognize a wide variety of substrates. Based on the observed wavelengths of maximum fluorescence emission from various CaM–target complexes, altering the chemical nature of the Met side chains has distinct effects on the local Trp environment of the target proteins. In our work, it appears that there are two major effects which decrease the magnitude of the observed blue-shift in Met analogue CaMs as opposed to WT-CaM complexes: stabilization of the excited state by selenium atoms through dipolar interactions; and a lack of Trp rigidity imposed by steric restrictions in ethionine (and sometimes norleucine) as compared with Met. In addition, the results confirm previous observations that quenching of Trp fluorescence intensity in CaM–target complexes is proportional to the atomic size of the atoms in place of Met, as the effectiveness of quenching proceeds Se > S > C. These results highlight the complexity of the interaction between CaM and its target proteins. The advantage of our approach is the relative simplicity in obtaining insight as to the role of Met in CaM–target binding; however, our understanding of the binding modes, and the nature of interaction between Met side chains and Trp is still lacking, and may possibly only be conclusively understood in the context of obtaining additional detailed three-dimensional structures through X-ray or NMR methods.

Our studies reveal that the two peptides derived from caldesmon, in particular CaD B, interact in a different manner with CaM. In part this must be related to the presence of a negatively charged Glu residue, immediately adjacent to the Trp, precluding it from completely entering the hydrophobic pocket(s) of CaM. Other CaM-binding domains are generally devoid of such proximal negatively charged residues, and are typically composed of hydrophobic and basic residues. We note that the disposition of the basic residues around the Trp in CaD A and CaD B peptides is different, possibly contributing to their differing affinities and binding characteristics to CaM.


    Acknowledgments
 
We are indebted to Dr M.M.Moloney for the use of his spectrofluorometer for steady state measurements. We would also thank Dr D.McKay for amino acid analysis. This work was supported by an operating grant from the National Sciences and Engineering Research Council of Canada (NSERC). A.M.W. would like to thank NSERC for his studentship. H.J.V. holds a Scientist Award from the Alberta Heritage Foundation for Medical Research (AHFMR).


    Notes
 
1 To whom correspondence should be addressed: email: vogel{at}ucalgary.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alix,J.-H. (1982) Microbiol. Rev., 46, 281–295.[ISI]

Babu,Y.S., Bugg,C.E. and Cook,W.J. (1988) J. Mol. Biol., 204, 191–209.[ISI][Medline]

Barabato,G., Ikura,M., Kay,L.E., Pastor,R.W. and Bax.A. (1992) Biochemistry, 31, 5269–5278.[ISI][Medline]

Blumenthal,D.K., Takio,K., Edelman,A.M., Carbonneau,H., Titani,K, Walsh,K.A. and Krebs,E.G. (1985) Proc. Natl Acad. Sci. USA, 82, 3187–3191.[Abstract]

Bowen,H.J.M. (1953) Tables of Interatomic Distances and Configuration in Molecules and Ions. Chemical Society, London.

Callis,P.R. (1997) Methods Enzymol., 278, 113–150.[ISI][Medline]

Charbonneau,H., Kumar,S., Novack,J.P., Blumenthal,D.K., Griffin,P.R., Shabanowitz,J., Hunt,D.F., Beavo,J.A. and Walsh,K.A. (1991) Biochemistry, 30, 7931–7940.[ISI][Medline]

Chen,Y. and Barkley,M.D. (1998) Biochemistry, 37, 9976–9982.[ISI][Medline]

Clore,G.M., Bax,A., Ikura,M. and Gronenborn,A.M. (1993) Curr. Opin. Struct. Biol., 3, 838–845.[ISI]

Comte,M., Maulet,Y. and Cox,J.A. (1983) Biochem. J., 209, 269–272.[ISI][Medline]

Crivici,A. and Ikura,M. (1995) Annu. Rev. Biophys. Biomol. Struct., 24, 85–116.[ISI][Medline]

Edwards,R.A., Walsh,M.P., Sutherland,C. and Vogel,H.J. (1998) Biochem.J., 331, 149–152.[ISI][Medline]

Gellman,S.H. (1991) Biochemistry, 30, 6633–6636.[ISI][Medline]

Graether,S.P., Heinonen,T.Y.K., Raharjo,W.H., Jin,J.-P. and Mak,A. (1997) Biochemistry, 36, 364–369.[ISI][Medline]

Huber,P.A. (1997) Int. J. Biochem. Cell. Biol., 8/9, 1047–1051.

Huber,P.A.J., El-Mezgueldi,M., Grabarek,Z., Slatter,D.A. and Levine,B.A. (1996) Biochem. J., 316, 413–420.[ISI][Medline]

Ikura,M. (1996) Trends Biochem. Sci., 21, 14–17.[ISI][Medline]

Ikura,M., Clore,G.M., Gronenborn,A.M., Zhu,G., Klee,C.B. and Bax,A. (1992) Science, 256, 632–638.[ISI][Medline]

James,P., Vorherr,T. and Carafoli,E. (1995) Trends Biochem. Sci., 20, 38–42.[ISI][Medline]

Klee,C.B., Crouch,T.H. and Krinks,M.H. (1979) Proc. Natl. Acad. Sci. USA, 76, 6270–6273.[Abstract]

Koradi,R., Billeter,M. and Wüthrich,K. (1996) J. Mol. Graphics, 14, 51–55.[ISI][Medline]

Krebs,E.G. (1985) Proc. Natl Acad. Sci. USA, 82, 3187–3191.[Abstract]

Kuboniwa,H., Tjandra,N., Grzesiek,S., Ren,H., Klee,C.B. and Bax,A. (1995) Nature Struct. Biol., 2, 768–776.[ISI][Medline]

Lakowicz,J.R.(1983) Principles of Fluorescence Spectroscopy. Plenum Press, New York.

Marston,S.B., Fraser,I.D.C., Huber,P.A.J., Pritchard,K., Gusev,N.B. and Torok,K. (1994) J. Biol. Chem., 269, 8134–8139.[Abstract/Free Full Text]

Meador,W.E., Means,A.R. and Quiocho,F.A. (1992) Science, 257, 1251–1255.[ISI][Medline]

Meador,W.E., Means,A.R. and Quiocho,F.A. (1993) Science, 262, 1718–1721.[ISI][Medline]

Means,A.R., Van Berkum,M.F.A., Bagchi,I., Lu,K.P. and Rasmussen,C.D. (1991) Pharmacol. Ther., 50, 255–270.[ISI][Medline]

O'Neil,K.T. and DeGrado,W.F. (1990) Trends Biochem. Sci.,15, 59–64.[ISI][Medline]

O'Neil,K.T., Wolfe,H.R.,Jr Erickson-Viitanen,S. and DeGrado,W.F. (1987) Science, 236, 1454–1456.[ISI][Medline]

Ogata,C.M. (1998) Nat. Struct. Biol. 5 supp., 638–640.[ISI][Medline]

Ohno,K, Matsuura,H. and Murata,H. (1980) J. Mol. Struct., 66, 45–64.[ISI]

Roth,S.M., Schneider,D.M., Strobel,L.A., VanBerkum,M.F.A., Means,A.R. and Wand,A.J. (1991) Biochemistry, 30, 10078–10084.[ISI][Medline]

Scaloni,A., Miraglia,N., Orru,S., Amodeo,P., Motta,A., Marino,G. and Pucci,P. (1998) J. Mol. Biol., 277, 945–958.[ISI][Medline]

Seeholzer,S.H., Cohn,M., Putkey,J.A., Means,A.R. and Crespi,H.L. (1986) Proc. Natl. Acad. Sci. USA, 83, 3634–3638.[Abstract]

Seeholzer,S.H., Cohn,M., Wand,A.J., Crespi,H.L., Putkey,J.A. and Means,A.R. (1987) In Calcium-binding Proteins in Health and Disease. Academic Press, New York, pp. 360–371.

Torok,K., Cowley,D.J., Brandmeier,B.D., Howell,S., Aitken,A. and Trentham,D.R. (1998) Biochemistry, 37, 6188–6198.[ISI][Medline]

Van der Spoel,D., de Groot,B., Hayward,S., Berendsen,H.J.C. and Vogel,H.J. (1996) Protein Sci., 5, 2044–2053.[Abstract/Free Full Text]

Vogel,H.J., Lindahl,L. and Thulin,E. (1983) FEBS Lett., 157, 241–246.[ISI]

Vogel,H.J. (1994) Biochem. Cell. Biol., 72, 357–376.[ISI][Medline]

Vogel,H.J. and Zhang,M. (1995) Mol. Cell. Biochem., 149/150, 3–15.[ISI]

Waltersson,Y., Linse,S., Brodin,P. and Grundstrom,T. (1993) Biochemistry, 32, 7866–7871.[ISI][Medline]

Wang,E., Zhuang,S., Kordowska,J., Grabarek,Z. and Wang,C.-L.A. (1997) Biochemistry, 36, 15026–15034.[ISI][Medline]

Yuan,T. and Vogel,H.J. (1999) Protein Sci., 8, 113–121.[Abstract]

Yuan,T., Weljie,A.M. and Vogel,H.J. (1998) Biochemistry, 37, 3187–3192.[ISI][Medline]

Yuan,T., Ouyang,H. and Vogel,H.J. (1999a) J. Biol. Chem., 274, 8411–8420.[Abstract/Free Full Text]

Yuan,T., Walsh,M.P., Sutherland,C., Fabian,H. and Vogel,H.J. (1999b) Biochemistry, 38, 1446–1455.[ISI][Medline]

Zhang,M. and Vogel,H.J. (1993) J. Biol. Chem., 268, 22420–22428.[Abstract/Free Full Text]

Zhang,M. and Vogel,H.J. (1994a) Biochemistry, 33, 1163–1171.[ISI][Medline]

Zhang,M. and Vogel,H.J. (1994b) J. Mol. Biol., 239, 545–554.[ISI][Medline]

Zhang,M., Li,M., Wang,J.H. and Vogel,H.J. (1994) J. Biol. Chem., 269, 15546–15552.[Abstract/Free Full Text]

Zhang,M., Tanaka,T. and Ikura,M. (1995) Nature Struct. Biol., 2, 758–767.[ISI][Medline]

Zhan,Q., Wong,S.S. and Wong,C.-L.A (1991) J. Biol. Chem., 266, 21810–21814.[Abstract/Free Full Text]

Zhou,N., Yuan,T., Mak,A.S. and Vogel,H.J. (1997) Biochemistry, 36, 2817–2825.[ISI][Medline]

Received May 7, 1999; revised November 5, 1999; accepted November 5, 1999.