Structural background of cyclodextrin–protein interactions

F.L. Aachmann, D.E. Otzen, K.L. Larsen and R. Wimmer1

Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark

1 To whom correspondence should be addressed. e-mail: rw{at}bio.auc.dk


    Abstract
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 Abstract
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 Materials and methods
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 Discussion
 References
 
Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological applications. We have studied the interaction between ß-cyclodextrin and four non-carbohydrate-binding model proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6 and insulin SerB9Asp by NMR spectroscopy at varying structural detail. We demonstrate that the interaction of ß-cyclodextrin and our model proteins takes place at specific sites on the protein surface, and that solvent accessibility of those sites is a necessary but not compelling condition for the occurrence of an interaction. If this behaviour can be generalized, it might explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically ‘masked’ by cyclodextrin), alteration of function (if residues involved in function are ‘masked’ by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be related to the particular structure of this protein.

Keywords: cyclodextrin–protein interaction/inclusion complex formation/NMR


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A large and growing number of peptide and protein drugs are used in therapy. The application of many of those, however, is hindered by unfavourable solubility, aggregation or instability. Also in biotechnological applications, the use of proteins is often severely hampered by their tendency to aggregate irreversibly. Many attempts have been undertaken to overcome these problems by using cosolutes (in the pharmacological world also often called adjuvants). Of these, cyclodextrins have in many cases proved useful (Irie and Uekama, 1999Go). Cyclodextrins are circular oligosaccharides composed of {alpha}-(1->4)-linked {alpha}-D-glucosyl units (Szejtli, 1998Go). The most common and industrially utilized cyclodextrins are {alpha}-, ß- and {gamma}-cyclodextrin, which consist of six, seven and eight glucosyl units. Larger cyclodextrins are known (Larsen, 2002Go), but are currently of low industrial relevance. Large cyclodextrins can be used for refolding of proteins (Machida et al., 2000Go). Cyclodextrins can be visualized as toroidal, hollow, truncated cones with hydrophilic rims crowned by OH-6 primary groups on the narrow rim and OH-2 and OH-3 secondary hydroxyl groups on the wide rim. In contrast, the internal cavity is hydrophobic, lined with H-3, H-5 and H-6 hydrogens and O-4 ether oxygens (Szejtli, 1998Go). This and the lack of structural freedom lead to a unique molecular structure that is water soluble but admits hydrophobic moieties, such as small organic molecules or hydrophobic residues on the protein surface. Cyclodextrins’ ability to sequester hydrophobic moieties on protein surfaces makes them valuable in two areas. First, within drug delivery, both for proteins (Irie and Uekama, 1999Go) and small molecules (Szejtli and Osa, 1996Go). For example, hydroxypropyl-ß-cyclodextrin enhances the affinity and antagonist potency of a neuromedin B receptor antagonist, the peptide PH 168368, by increasing its solubility (Ryan et al., 1999Go), while dimethyl-ß-cyclodextrin increases the nasal absorption of leucine enkephalin (Agu et al., 2002Go). Secondly, for stabilization of proteins against aggregation, thermal denaturation and degradation. Thus, cyclodextrins increase the shelf-life of therapeutic proteins, such as growth hormones (Hagenlocher and Pearlman, 1989Go; Brewster et al., 1991Go; Charman et al., 1993Go) and insulin (Banga and Mitra, 1993Go). Numerous reports of cyclodextrins inhibiting or slowing down the aggregation of proteins have appeared in the literature (Sigurjonsdottir et al., 1999Go; Leung et al., 2000Go; Otzen et al., 2002Go). Cyclodextrins have also been suggested to act as ‘chaperone mimics’ by enhancing protein refolding from denatured or even aggregated states (Karuppiah and Sharma, 1995Go; Akiyoshi et al., 1999Go; Bär et al., 2000Go; Sharma and Sharma, 2001Go; Dong et al., 2002Go). Hydroxypropyl-ß-cyclodextrin inhibits the spray-drying-induced inactivation of ß-galactosidase (Branchu et al., 1999Go), while a carboxymethyl- cyclodextrin increases the thermostability and emulsifying activity of ß-lactoglobulin (Hattori et al., 2000Go). Depending on the cyclodextrin and the peptide, both stabilization and destabilization against proteolytic and chemical degradation have been reported (Sigurjonsdottir et al., 1999Go; Koushik et al., 2001Go).

The origin of these phenomena has been suggested to be a specific interaction between cyclodextrin and hydrophobic amino acids (Cooper, 1992Go; Horsky and Pitha, 1994Go; Breslow et al., 1998Go). Koushik et al. (2001Go) showed that the UV and fluorescence spectroscopy behaviour of the D-tryptophan residue of deslorelin, a peptide drug, was altered by the addition of cyclodextrin. They also determined a binding constant and a reaction enthalpy by isothermal titration calorimetry. Matsubara et al. (1997Go) showed by UV, circular dichroism and NMR spectroscopy that dimethyl-ß-cyclodextrin incorporates the aromatic side chains of L-tryptophan and L-tyrosine of buserelin acetate (a peptide drug), into its cavity. The interaction of human growth hormone with maltosyl-ß-cyclodextrin was shown to cause large shifts of the 1H-NMR signals of the tyrosine residues (Uekama et al., 1998Go). NMR has been used in conjunction with fluorescence spectroscopy and kinetics to prove and quantitate the inclusion complex formation of ß-cyclodextrin and aromatic amino acid residues of human growth hormone (Otzen et al., 2002Go). A corollary of the affinity for aromatic amino acids is that cyclodextrins shift the thermodynamic equilibrium towards the unfolded state, simply because the unfolded state offers more potential binding sites than the native state (Cooper, 1992Go).

Several crystal structures of carbohydrate-binding proteins in complex with cyclodextrins are available in the PDB database (Berman et al., 2000Go). Most of them indicate interactions between cyclodextrins and aromatic amino acids, though soybean ß-amylase highlights an unusual contact between the isopropyl group of a leucine side chain and the wide rim of the cyclodextrin cavity (Table I).


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Table I. Summary of protein–cyclodextrin interactions in complexes of known structure retrieved from the PDB database (Berman et al., 2000)
 
In contrast to the considerable structural insights into the interactions between cyclodextrins and carbohydrate-binding proteins, there are only a few reports on contacts between cyclodextrins and proteins that do not specifically interact with carbohydrates (Matsubara et al., 1997Go; Uekama et al., 1998Go; Koushik et al., 2001Go). No structural information on such interactions has been reported so far. To obtain more general and structural information on protein–cyclodextrin interactions without functional bias, we have undertaken a structural study of the binding of ß-cyclodextrin to four different non-carbohydrate-binding proteins, namely ubiquitin, chymotrypsin inhibitor 2, S6 and the drug insulin [as the mutant SerB9Asp whose aggregation state can be controlled by pH (Brange et al., 1988Go)]. The three latter proteins have been used as model systems in protein folding and stability studies. The structures of all four proteins are known (Table II).


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Table II. Proteins used in this study
 
The insulin mutant was chosen because of its favourable aggregation properties. Whereas wild-type insulin exhibits heavy aggregation at concentrations in the millimolar regime, which are needed for NMR studies, the SerB9Asp mutant is essentially monomeric at pH ~>7 and dimeric at lower pH values (Brange et al., 1988Go; Kristensen et al., 1991Go; Sørensen and Led, 1994Go). We found this behaviour consistent with our own observations, where inter-domain monomer–monomer nuclear Overhauser effects (NOEs) could be observed at pH* 1.5, but not at pH* 6.8 and above. Since some of the presumed binding sites for ß-cyclodextrin are buried by dimerization, but solvent accessible in the monomeric form, this mutant was ideal for our study.

We have chosen to use NMR spectroscopy for these investigations because it is capable of providing atomic level information about complex supramolecular systems. Moreover, the NOE with its dependence on the structure of the system provides structural information on the inclusion complex(es) formed.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Human insulin with a point mutation SerB9Asp was kindly provided by Novo Nordisk, Bagsvaerd, Denmark. CI2 (Jackson et al., 1993Go) and S6 (Otzen et al., 1999Go) were purified as described previously. Ninety percent pure bovine ubiquitin was purchased from Sigma-Aldrich, Vallensbæk Strand, Denmark. Pharmaceutical grade {alpha}-, ß- and {gamma}-cyclodextrin were obtained from Wacker-Chemie GmbH, Burghausen, Germany. Thirty five percent DCl (D 99.5%), 99.9 and 99% pure D2O were from Larodan Fine Chemicals AB, Malmö, Sweden. Stock solutions of 0.5 M NaD2PO4 and 0.5 M Na2DPO4 were prepared by drying the powders to reduce water signals and subsequently dissolving in D2O and drying. Phosphate buffer is used as the buffer system in the sample, since it does not show an interaction with cyclodextrin (Cramer et al., 1967Go). {alpha}-, ß- and {gamma}-cyclodextrin were treated in the same way to reduce the residual water signal.

Only uncorrected pH meter readings are given in this paper. If the solvent was D2O, this is indicated by designating it pH* instead of pH.

NMR spectroscopy

All 1H-NMR spectra were recorded at 600 MHz on a BRUKER DRX600 spectrometer equipped with a 5 mm xyz-grad TXI(H/C/N) probe. The NMR data were processed with the BRUKER XwinNMR Ver. 2.5 software and the spectral analysis was done with XEASY Ver. 1.3.13. (Bartels et al., 1995Go).

Insulin SerB9Asp. The insulin SerB9Asp NMR sample was prepared by dissolving insulin SerB9Asp to a final concentration of 0.6 mM in 550 µl of 99.9% D2O with 50 mM phosphate buffer measured to a pH* of 6.8 and 1.5. NOESY spectra with 100 ms mixing time were conducted on both samples, a DQF-COSY of the sample at pH* 6.8 was recorded. Afterwards, ß-cyclodextrin was added to a final concentration of 12 mM after the recording was completed on the sample without ß-cyclodextrin. NOESY spectra with 100 ms mixing time were conducted on insulin and ß-cyclodextrin with a pH* of 10.6, 10.1, 9.1, 6.8 or 1.5 at 298 K.

CI2. The CI2 stock solution was freeze-dried and resuspended in 99% D2O and freeze-dried again in order to reduce the water signal in the spectra. The unfolded CI2 NMR sample was prepared from 2 mM CI2 in 350 µl of 99.9% D2O with 120 mM DCl measured to a pH* of 0.5. ß-Cyclodextrin was added to a final concentration of 12 mM in the unfolded CI2 sample after recording was completed on the sample without ß-cyclodextrin. The folded CI2 NMR sample was prepared as 2 mM CI2 in 350 µl of 99.9% D2O with 50 mM phosphate buffer measured to a pH* of 5.9. When the recording of the sample of CI2 without ß-cyclodextrin was completed, ß-cyclodextrin was added to a final concentration of 12 mM.

NOESY spectra with 90 ms mixing time were conducted to detect any intermolecular interactions between cyclodextrin and CI2.

Ubiquitin. The ubiquitin NMR sample was prepared from 5.2 mg of ubiquitin dissolved in 550 µl of 99% D2O with 50 mM phosphate buffer, pH* 5.8 or pH* 2.6. ß-Cyclodextrin was added to a final concentration of 12 mM after the recording on the sample without ß-cyclodextrin was completed. NOESY spectra with 100, 250 and 500 ms mixing time were conducted to detect any intermolecular interactions between ß-cyclodextrin and ubiquitin at pH* 5.78, 303 K. NOESY spectra with 250 ms mixing time were conducted on ubiquitin and ß-cyclodextrin with a pH* of 2.6 at 303, 323 and 340.5 K.

S6. Samples of 1 mM S6 were prepared in a deuterated 20 mM MES buffer/50 mM NaCl at pH* 7.4. NOESY spectra with 100 ms mixing time were conducted. Afterwards, ß-cyclodextrin was added to a concentration of 10.6 mM and a NOESY spectrum with identical parameters was recorded.

The same procedure was applied to samples of 0.6 mM S6 in 10 M deuterated urea at pH* 7.4. All S6 spectra were recorded at 298 K.

Stopped-flow fluorescence

The kinetics of CI2’s refolding from the acid-denatured state were followed on an Applied Photophysics SX18MV stopped-flow system (Applied Photophysics, Leatherhead, Surrey, UK), using the large decrease in fluorescence of the single tryptophan upon refolding. Excitation was at 280 nm and emission was followed above 320 nm using a cut-off filter. The total mixing volume of the apparatus was 150 µl, and the measurements were performed by mixing CI2 and buffer (1:10, v/v) solutions at 288 K. The measurements were performed with 55 µM CI2 in 20 mM HCl at pH 1.7 mixed with 100 mM phosphate buffer at pH 7.28 to a final concentration of 5 µM CI2 and a cyclodextrin concentration of 0, 1, 2, 4, 8, 16, 32 and 64 mM for {alpha}- or {gamma}-cyclodextrin and of 0, 1, 2, 4, 8 and 10.8 mM for ß-cyclodextrin. Each measurement was repeated between three and seven times. Data were fitted to a double exponential decay, in which the phase with the larger amplitude and faster rate is the major refolding phase, while the slower phase is associated with proline isomerization (Jackson and Fersht, 1991Go).


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 Abstract
 Introduction
 Materials and methods
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 References
 
Insulin

The NOESY spectra of dimeric insulin SerB9Asp in the presence of ß-cyclodextrin at pH* 1.5 shows cross-peaks between H-3 and H-5 of ß-cyclodextrin and aromatic insulin protons (Figure 1B). Based on the assignment of Kristensen et al. (1991Go), these could be identified as belonging to the H{delta} and H{epsilon} protons of TyrA14. Additional cross-peaks occurring in the same region of the NOESY spectra are due to NOEs between different monomeric subunits. These peaks could be assigned based on Jørgensen et al. (1992Go) (Figure 1A).



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Fig. 1. Region of NOESY spectra where the interaction between cyclodextrin and insulin SerB9Asp can be seen: (A) without cyclodextrin at pH* 1.5, where the protein exists as a dimer which gives rise to intermonomer NOEs in the same region of the spectrum, (B) as (A) but with cyclodextrin present, (C) without cyclodextrin at pH* 6.8, (D) with cyclodextrin at pH* 6.8, (E) with cyclodextrin at pH* 9.1 and (F) with cyclodextrin at pH* 10.1.

 
The NOESY spectrum of monomeric insulin SerB9Asp in the presence of ß-cyclodextrin at pH* 6.8 reveals a multitude of interactions between several aromatic protons of the insulin and the H-3 and H-5 protons of the ß-cyclodextrin (Figure 1D). The cross-peaks at ~6.8 p.p.m. with H-3 and H-5 of cyclodextrin are assigned to H{epsilon} of both TyrA14 and TyrB16 based on the chemical shift list of Roy et al. (1990Go). The cross-peak at 7.11 p.p.m. with H-3 and H-5 of cyclodextrin is assigned to H{delta} of TyrA14 which is compatible with the assignment (Roy et al., 1990Go; Kristensen et al., 1991Go). The weak cross-peaks at 7.18 p.p.m. with H-3 and H-5 can be unambiguously assigned to H{delta} of PheB1. The remaining cross-peaks between H-3 and H-5 of ß-cyclodextrin and aromatic insulin protons could stem from H{delta} of TyrA19, H{delta} of TyrB16, H{epsilon} and H{zeta} of PheB1 and H{delta}, H{epsilon}, H{zeta} of PheB25. If there was an interaction between cyclodextrin and PheB24 or TyrB26, additional cross-peaks should be seen in separate spectral regions. The absence of any cross-peaks in these regions can be used to rule out any interaction of cyclodextrin with these two residues.

To resolve the ambiguity and to find the real interaction sites, the NOESY spectra were in addition recorded at pH* 9.1, 10.1 and 10.6. By raising the pH, the Tyr residues are deprotonated, which gives rise to a change in chemical shift. The nearby Phe residues will be affected indirectly, but not to the same extent. Reducing spectral overlap made it possible to assign the residues that interact with ß-cyclodextrin. The NOESY spectrum at pH* 9.1 shows more distinct cross-peaks between insulin SerB9Asp and ß-cyclodextrin (Figure 1E). The cross-peak at 6.83 p.p.m. with H-3 and H-5 of cyclodextrin is assigned to H{epsilon} of TyrA14 which is compatible with the assignment of Roy et al. (1990Go). The cross-peaks at 6.90 and 7.34 p.p.m. to H-5 and H-3 of ß-cyclodextrin fits to TyrB16 H{epsilon} and H{delta}, respectively. The cross-peaks around 7.28–7.29 p.p.m. to H-5 and H-3 of ß-cyclodextrin could still be assigned to both the aromatic protons of PheB25 and PheB1. Additionally, an interaction with TyrA19 cannot be ruled out based on this spectrum alone.

At pH* 10.1 the cross-peaks between the aromatic protons of PheB1 and PheB25 to H-5 and H-3 can be resolved. The changes in chemical shift for the individual aromatic protons correlate with the assignment from Roy et al. (1990Go). It can clearly be seen that both Phe residues in question show an interaction with cyclodextrin. The spectrum at pH* 10.6 supported the results obtained from the other spectra, but did not provide any additional information.

Another point of special interest is whether there are interactions between cyclodextrins and the aliphatic groups of leucine and isoleucine residues. No cross-peaks to ß-cyclodextrin outside the aromatic region were found in any of the NOESY spectra recorded. As the region in the spectra where cross-peaks between cyclodextrin protons and aliphatic protons would be expected is very crowded, this cannot be seen as an absolute proof for the absence of such interactions.

From our data we see that there are four interaction sites on the monomeric and one per monomeric subunit on the dimeric form of insulin. While we are unable to determine dissociation constants at these interaction sites, the larger number of interaction sites on the monomer compared with the dimer is consistent with the results of Lovatt et al. (Lovatt et al., 1996Go) who demonstrated, by calorimetric dilution data, that high concentrations of cyclodextrin increase the dissociation of insulin dimers to monomers. Their data show that cyclodextrin has at least two binding sites on the insulin monomer. Their data can be fitted to a 1:2 model with dissociation constants Kd,1 = 50–100 mM and Kd,2 > 150 mM. A direct comparison of their results with our results is problematic because we used ß-cyclodextrin, whereas they used {alpha}-cyclodextrin, but the interaction between aromatic amino acids and {alpha}- or ß-cyclodextrin, respectively, is similar in inclusion complex geometry and only marginally weaker for {alpha}-cyclodextrin (Connors, 1997Go; Aachmann, 2001Go).

Given that we identify four interaction sites on insulin, it is likely that the first Kd,1 is composed of several microscopic binding constants. However, our NMR data do not allow us to determine individual binding constants.

CI2. The 2D NOESY spectrum of unfolded CI2 in the presence of ß-cyclodextrin clearly shows an interaction between the H-3 and H-5 proton of the ß-cyclodextrin and the aromatic residue Trp5 in CI2 (Figure 2), assigned by using the random coil chemical shift for amino acids (Bundi and Wüthrich, 1979Go) and comparing the cross-peak pattern to that of the interaction of cyclodextrin with free tryptophan (data not shown). In the area of interest for the NOESY spectra of unfolded CI2 without ß-cyclodextrin there are no signs of interaction. The spectrum did not show an interaction between ß-cyclodextrin and tyrosine or phenylalanine. This is surprising because the association constant and the excess of ß-cyclodextrin should be enough for both tyrosine and phenylalanine to interact. One possible explanation for this is that the two residues are not sufficiently solvent exposed, which could be due to the presence of residual structure in the form of hydrophobic interactions. A number of ill-defined cross-peaks between aromatic protons and methyl protons in the NOESY spectrum of the unfolded CI2 with and without ß-cyclodextrin (Figure 3) would also corroborate this explanation. The residual structure need not involve residues far apart in sequence, but could be flickering local interactions that obstruct cyclodextrin access.



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Fig. 2. NOESY spectrum for 2 mM unfolded CI2 with 12 mM ß-cyclodextrin present in 120 mM DCl at pH* 0.5 and 288 K. (A) The cross-peaks between ß-cyclodextrin and Trp5 and (B) the whole spectrum. The dotted circle highlights some ill-defined cross-peaks between aromatic and methyl protons, which could indicate residual structure.

 


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Fig. 3. The observed rate constant (kobs) versus cyclodextrin concentration. KCD,d for {alpha}-cyclodextrin (· x ·) is estimated to 26.88 ± 3.28 (mM) and R2 = 0.9904. KCD,d for ß-cyclodextrin (· + –) is estimated to 18.94 ± 0.69 (mM) and R2 = 0.9989. KCD,d for {gamma}-cyclodextrin (– {Delta} –) is estimated to 47.72 ± 9.03 (mM) and R2 = 0.9589.

 
The spectrum of folded CI2 with ß-cyclodextrin does not show any sign of interaction between aromatic protons or other protons of CI2 and ß-cyclodextrin (data not shown).

Stopped-flow fluorescence

To estimate the degree of interaction between cyclodextrin and the pH-denatured state of CI2, we measured the protein’s folding kinetics at increasing cyclodextrin concentrations. The rate constant for the folding of CI2 declines with increasing cyclodextrin concentration (Figure 3). Assuming a dynamic equilibrium between unfolded CI2 bound to cyclodextrin and free unfolded CI2, the folding rate for CI2 can be described by the following equation, in which the observed rate kobs reflects the folding of free unfolded CI2:

where kobs (s–1) is the observed folding rate, KCD,d (M) is the dissociation constant for the cyclodextrin interaction with unfolded CI2, and kf (s–1) is the folding rate in the absence of cyclodextrin. By plotting the observed rate constant (kobs) versus cyclodextrin concentration, KCD,d can be determined. The estimated KCD,d values are 26.9 ± 3.3 mM for {alpha}-, 18.9 ± 0.7 mM for ß- and 47.7 ± 9.0 mM for {gamma}-cyclodextrin.

Ubiquitin. NOESY spectra with a mixing time of 100, 150 and 250 ms were conducted on ubiquitin and ß-cyclodextrin at 303 K, in order to detect any interactions. These spectra did not show any sign of interaction between ß-cyclodextrin and ubiquitin (data not shown). Attempts to raise the temperature and lower the pH in order to destabilize the folded form and thus increase the probability of finding a ubiquitin–cyclodextrin complex were not successful.

S6. Native S6 exhibited clear interactions between the aromatic regions of the protein and the cyclodextrin (Figure 4). Although no assignment is available, one can, on the basis of the signals, conclude that two aromatic side chains interact with cyclodextrin. From the structure of S6, Phe50 and Tyr97 can be identified to show a high solvent accessibility. The position of the NMR resonances interacting with cyclodextrin is consistent with one Phe and one Tyr residue, respectively. We also attempted to study the interaction between ß-cyclodextrin and urea-denatured S6. S6 does not denature at low pH and is not completely denatured even in 10 M urea, but a highly destabilized double mutant of S6 (Leu30Ala/Val37Ala) has a denaturation midpoint of ~4 M urea (data not shown). In 9 M urea, we can identify more interactions between Leu30Ala/Val37Ala and cyclodextrin than in the absence of urea.



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Fig. 4. Region of NOESY spectrum of S6 with ß-cyclodextrin. The cross-peaks in this region (except for the weak peak at 6.66/4.05 p.p.m.) are not present in the absence of ß-cyclodextrin (data not shown).

 
However, in addition to unfolding the protein, urea also reduces the cyclodextrin’s sequestering ability. The Kd of the Phe-CD increases from 6.9 mM in water to 188 mM in 6 M urea as determined by NMR diffusion measurements (Wimmer et al., 2002Go). This is probably due to urea’s ability to solvate aromatic residues. The practical consequence of this is that the signals become weaker.


    Discussion
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 Abstract
 Introduction
 Materials and methods
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 Discussion
 References
 
Here we have described a study of the binding of ß-cyclodextrin to four different non-carbohydrate-binding proteins with known structures: insulin SerB9Asp, CI2, ubiquitin and S6. It has been demonstrated that the interaction between ß-cyclodextrin and these model proteins takes place at specific locations on the protein. If these findings can be generalized, they might explain the wide variety of effects of cyclodextrins on protein preparations that have been observed.

Based on the investigation of the insulin–ß-cyclodextrin interaction, it can be seen that ß-cyclodextrin interacts with specific solvent exposed amino acid residues in the monomer–monomer interaction domain of insulin. By placing a hydrophilic ‘cap’ on aromatic residues exposed in the monomer but buried in the dimer interface, cyclodextrins sterically block dimer formation; they also short-circuit the burial of hydrophobic surface area that would otherwise stabilize the dimer (Lovatt et al., 1996Go). Enhanced adsorption of insulin observed in pharmacological applications (Merkus et al., 1991Go; Irie et al., 1992Go; Watanabe et al., 1992aGo,b; Schipper et al., 1993Go) could be explained by our results, in that it is easier for the monomeric form of insulin to cross the mucosal linings than for the oligomeric form. It has, however, also been proposed that the effect might be due to the membrane structure being altered by cyclodextrins (Irie et al., 1992Go).

Can possible interaction sites be identified through the water-accessible surface area? The water-accessible surface area for the monomeric insulin SerB9Asp has been calculated by MOLMOL (Koradi et al., 1996Go). Table II shows that the water accessibility of the aromatic rings of TyrA19, PheB24 and PheB26 is low and therefore, these residues are less likely to interact with ß-cyclodextrin. The aromatic ring protons of residues TyrA14, PheB1, PheB25 and TyrB16 are to a large extent solvent exposed, and they should be able to interact with ß-cyclodextrin. Since this is in total agreement with the NMR data, one might speculate that every solvent-exposed residue—also in other proteins—is an interaction site.

However, data for the dimer reveal a more differentiated picture: dimer formation involves residues B8, B9, B12, B13, B16 and B23–B28, which means that one should expect that only TyrA14 and PheB1 can interact with ß-cyclodextrin when insulin is dimeric (Baker et al., 1988Go). Nevertheless, only TyrA14 appears to be interacting with ß-cyclodextrin in dimeric insulin judging from the obtained NMR results.

One should be wary of making detailed conclusions about structural features of a protein surface from a PDB file alone because the dynamics of distinct residues can strongly interfere with their ability to form complexes with cyclodextrin, yet it is not reflected in the static structure. An example for this could be PheB1 of the insulin SerB9Asp dimer, which appears to have a high solvent accessibility in the static structure, but does not show any interaction with cyclodextrin. Another example is the tyrosine side chain of folded CI2 which does not show an interaction with ß-cyclodextrin although some residues in insulin with a lesser solvent accessibility do.

The water-accessible surface areas of the aromatic side chains of ubiquitin are low (Table II and Figure 5), which can explain why no interactions with cyclodextrin were detected under a range of experimental conditions. Visual inspection of the ubiquitin structure (Figure 5) could, however, mislead one to believe that there might be at least one interaction with cyclodextrin.



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Fig. 5. Backbone of the model proteins insulin SerB9Asp (A), ubiquitin (B) and S6 (C), with the aromatic residues highlighted. Residues that were found not to interact with cyclodextrin are highlighted in red; those that interact are highlighted in green. The solvent-accessible surface part of the corresponding residues is shown in the colour of the residue. The correlation between solvent accessibility and interaction with cyclodextrin can clearly be seen.

 
S6 behaves regularly, too, as a combined inspection of water-accessible surface area and NOESY NMR spectra reveals: only two out of eight aromatic residues are solvent exposed (Table II), and only two are found to interact with cyclodextrins (Figures 4 and 5).

Our data clearly confirm that ß-cyclodextrin can bind to solvent-accessible aromatic side chains on the surface of proteins. But how likely is one to find exposed aromatic residues? Few tryptophan rings are completely exposed (Samanta et al., 2000Go), and the partially buried tryptophan ring of S6 does not interact with cyclodextrin. Apparently, an aromatic side chain needs substantial surface exposure to interact with cyclodextrin. Interestingly, tryptophan and tyrosine are the only hydrophobic residues which are found with equal frequency in the interior and on the surface of proteins (Tsai et al., 1997Go). Energetic hot spots, which contribute significantly to the association energy in protein complexes, are also enriched in tryptophan and tyrosine (Bogan and Thorn, 1998Go). Thus, cyclodextrins are likely to encourage the dissociation of protein complexes.

Conclusion

We have presented detailed data on the interaction between ß-cyclodextrin and insulin. We have data on S6 and the unfolded form of CI2 that reveal the same type of interaction between the protein and ß-cyclodextrin. In all these cases, ß-cyclodextrin admits a fairly solvent-exposed aromatic side chain into its cavity. Some of the crystal structures reported in Table I show the same interaction pattern. The lesser detailed studies on the interaction between peptide drugs and cyclodextrins, that are reported in the Introduction (Matsubara et al., 1997Go; Uekama et al., 1998Go; Koushik et al., 2001Go; Otzen et al., 2002Go), point in the same direction. Therefore, it is tempting to speculate that this is the predominant mode of interaction between proteins and cyclodextrins. If it is, it can explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically ‘masked’ by cyclodextrin), alteration of function (if residues involved in function are ‘masked’ by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be given by the particular structure of this protein.

This knowledge might be used to engineer ‘cyclodextrin-binding sites’ in the form of highly exposed aromatic side chains into proteins that for some reason or other need to be stabilized against aggregation, degradation or temporarily inactivated. The generally high dissociation constants (in the millimolar regime) are very useful with respect to technical utilization: while cyclodextrins in high concentrations can be present as long as their effect is desired (e.g. under storage), they can easily be diluted out before application of the enzyme, if necessary.


    Acknowledgements
 
We wish to thank Helle B.Olsen from Novo Nordisk A/S for helpful discussions on structure and assignment of the insulin mutant as well as providing the protein samples. F.L.A. wishes to acknowledge a Novo Scholarship and financial support from Novo Nordisk A/S. The NMR laboratory at Aalborg University is supported by the SparNord Foundation.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 30, 2003; revised October 27, 2003; accepted October 30, 2003





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