# Design of Beta-Lactoglobulins with Increased Affinities for Retinol and Palmitic Acid †

Damien Jayat, Jean-Charles Gaudin, Michèle Dalgalarrondo, Yvan Choiset, Jean-Marc Chobert, Vinh Tran, Carl Holt, Thomas Haertlé

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Damien Jayat, Jean-Charles Gaudin, Michèle Dalgalarrondo, Yvan Choiset, Jean-Marc Chobert, et al.. Design of Beta-Lactoglobulins with Increased Affinities for Retinol and Palmitic Acid †. 2005. [hal-04394773](https://hal.science/hal-04394773v1)

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Design of β-Lactoglobulins with Increased
Affinities for Retinol and Palmitic Acid†

† This work was supported by the Région Pays de la Loire and INRA for the Ph.D. fellowship awarded to D.J. and by the British Council and French Ministry of Foreign Affairs in the frame of Franco-British Alliance project 03039WF and 08780SD. L.S. and C.H. were also supported by a grant from the Biotechnology and Biological Sciences Research Council.

Damien Jayat‡, Jean-Charles Gaudin‡, Michèle Dalgalarrondo‡, Yvan Choiset‡, Jean-Marc Chobert‡, Vinh Tran§, Carl Holt‖, Lindsay Sawyer⊥, and Thomas Haertlé**

‡ Institut National de la Recherche Agronomique, BIA-Fonctions et Interactions des Protéines Laitières, rue de la Géraudière, B.P. 71627, 44316 Nantes Cedex 3, France

§ Université de Nantes, UMR CNRS 6204 Biotechnologie, Biocatalyse et Biorégulations, 2 rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France

‖ Hannah Research Institute, Ayr, KA6 5HL, U.K.

⊥ Institute of Cell and Molecular Biology, The University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR, U.K.

Running title: Increase of β-lactoglobulin affinity for two ligands

* Corresponding author. Tel. + 33 (0)2.40.67.50.91 ; Fax . + 33 (0)2.40.67.52.44 ; e-mail:
[haertle@nantes.inra.fr](mailto:haertle@nantes.inra.fr)
¹ Abbreviations: BLG, β-lactoglobulin; PA, palmitic acid; H-bond, hydrogen bond; PDB, Protein Data Bank; SDS-PAGE, Sodium Dodecyl-Sulfate Poly-Acrylamide Gel Electrophoresis; RP-HPLC, Reversed-Phase High-Performance Liquid Chromatography; BCA, Bicinchoninic Acid; WT, Wild-Type; ESI, Electron-Spray Ionization; CD, Circular Dichroism.
ABSTRACT: The cow's milk protein β-lactoglobulin (BLG) is able to bind *in vitro* and *in vivo* a large variety of small, hydrophobic molecules. This property can be exploited for the transport of therapeutic compounds that are insoluble, partially toxic and/or that need to be precisely targeted. As a first step, the mechanisms that govern the strength and the specificity of BLG - ligand interaction need to be identified. Here we report the site-directed mutagenesis of bovine BLG based on the crystal structure of the protein complex with its two main ligands, retinol and palmitic acid. The modifications were chosen after molecular modeling simulations and concerned residues K69 and A86, both located at the entry of the main ligand-binding site. BLG affinity for retinol and palmitic acid was respectively increased up to 5.5 (K69R-A86Q substitution) and 10-fold (K69N-A86Q substitution). Most of the experimental results correlated with the simulations. Several parameters influencing BLG - ligand interaction were pointed out, such as the flexibility of the ligand or the geometry of the hydrogen-bond network between BLG and the ligand polar group. These results increase our knowledge about ligand binding to BLG, and constitute an important step towards further mutagenesis experiments carried out in order to increase BLG specificity for biologically important ligands.
Bovine BLG¹ is the major whey protein of cow's milk. It is a small (18.4 kDa) globular protein composed of 162 amino acids. Solution of the BLG crystal structure (1) revealed that it belongs to the "lipocalin" family, a group of proteins involved in the transport of hydrophobic molecules (2). BLG is composed of eight anti-parallel β-strands organized as a β-barrel, whose inside forms a hydrophobic pocket or "calyx" (Fig. 1). A 3-turn α-helix lies along the outer side the barrel, followed by a ninth short β-strand. BLG exists as a homodimer in physiological conditions, the dimer interface being composed of the AB loop and strand I of each monomer (3).

In solution, BLG is able to bind a large variety of small hydrophobic molecules, mainly retinoids (4-7), fatty acids (8-10), triglycerides (11), sterols (6, 12) and aromatic compounds (13, 14). Its affinity for these molecules is variable, with dissociation constants ($K_d$) ranging from $10^{-2}$ to $10^{-8}$ M. The highest affinities have been measured for retinol and long-chain fatty acids such as palmitic acid (PA, Ref 15).

The identity and number of ligand-binding sites on BLG have been debated for a long time. Results from many *in vitro* studies were interpreted in terms of a unique site located inside the β-barrel (4, 9, 12, 16-21). This model was confirmed by crystal structure of BLG complexed with 12-bromo-dodecanoic acid (22), PA (23), retinol and retinoic acid (24). Moreover, BLG with the 3-turn α-helix removed has the same affinity for retinal (25) or *cis*-parinaric acid (26) as the intact protein, and retinol interaction with a lysyl residue located at the entry of the β-barrel has been demonstrated in solution (27). On the other hand, some experiments have suggested the existence of additional binding sites for fatty acids (8, 28, 29) and retinoids (30, 31) or have shown that BLG was able to bind simultaneously and at different sites these two families of ligands (32).
Taken together, these studies suggest that BLG possesses i) one strong ligand-binding site for retinol and fatty acids per protein monomer, located inside the hydrophobic calyx, ii) one or several secondary binding sites, located on the outer surface of the protein.

The ability of BLG for ligand binding may be related to its biological function(s), which remain poorly defined. However, the presence of one strong binding site located inside the protein could be exploited in delivery systems enhancing the control of drug bioavailability. For example, there is a need for protecting labile compounds from degradation, for solubilizing lipophilic molecules, or for targeting drugs to their specific destination with the highest precision (33). This targeting can be mediated by monoclonal antibodies (34, 35) or by specific carrier proteins such as albumin, lactoferrine or streptavidin (36-38).

Bovine BLG is a good candidate for drug transport, particularly for the solubilization, protection and/or targeting of hydrophobic compounds. Interestingly, BLG is resistant to gastric proteolysis (by pepsin in particular) hence it is able to pass intact into the intestine. However, the natural affinity of BLG for all tested ligands is too weak, since $K_d$ lower than $10^{-10}$ or $10^{-11}$ M are best suited design transport (34). Thus, the need for improving BLG affinity and/or specificity for ligands of interest through mutagenesis has been pointed out by several authors (33, 39-42).

Ligand binding in the BLG calyx is mediated by two types of interaction. First, there are hydrophobic interactions between the aliphatic chain of the ligands and the side-chains of residues located inside the barrel. Consequently, BLG affinity increases with the ligand hydrophobicity (8, 14, 19). Second, polar bonds occur between the ligand head-group and the side-chains of residues located at the entry of the calyx. For example, crystal structure analyses have suggested that PA interacts with residues K60 and K69 (23), while retinol interacts with residue E62 (24). However, precise information on the associations that
determine the strength of BLG - ligand interaction, and more particularly the electrostatic and
H-bond networks, are still lacking.

In this study we report the production and characterization of BLG mutants designed
to increase BLG affinity for retinol and PA. The substitutions concerned residues located at
the entry of the binding site and led to a modification of the polar interactions between BLG
and the two ligands. Our results have identified several parameters that influence BLG
affinity for hydrophobic molecules.

## EXPERIMENTAL METHODS

*Molecular modeling* – Simulations were performed using Insight II, Biopolymer and Discover softwares from Accelrys Inc. (San Diego, CA). The structures of BLG complexes with retinol (PDB coordinates 1GX8) and PA (PDB 1B0O) were used as templates. Energetic minimization of the structures was carried out by systematic analysis of i) the ligands’ polar groups, through modifications of the C1-C2 and C2-C3 dihedral angles and rotations around the carbon carrying the polar group, and ii) the amino acid residues located in the polar groups’ vicinity, by testing all possible side-chain orientations. The residues considered were K60, E62, K69, A86, L87 and N88 (Fig. 1). All combinations of side-chain and ligand orientations were tested by series of 10,000 to 20,000 iterations of energy minimization, first with the protein backbone fixed. The structure was progressively relaxed by allowing movement of the CD [residues 61-66] and EF [residues 85-90] loops (Fig. 1), and finally with the entire protein relaxed. The same maximum derivative criterion was used to stop the minimizations. Refined structures were obtained for each solution tested after 5-10 cycles of manual repositioning followed by energy minimization. One solution corresponding to a local energetic minimum was identified for both the BLG + retinol and BLG + PA structures.
Substitutions were tested for residues K69 and A86, which were substituted by ionisable or polar residues that could be accommodated in the BLG structure without steric hindrance (D, E, N, Q, H, K, R or Y). For each substitution, a systematic analysis was carried out as described for the native structures. The total energy of the protein – ligand complex (E<sub>tot</sub>) was optimised to take into account the necessary adaptation of both partners at the interface. Then, the interaction energy (IE) was derived from the formula $IE = E_{tot} - E_p - E_l$, where E<sub>p</sub> and E<sub>l</sub> are the energies of the protein and the ligand, respectively, and which were calculated separately. Clearly, this protocol (optimisation with force field CFF91 and without explicit water molecules) produces a unique solution, considered to correspond to the lowest energy one, but it should be noted that no account was taken of entropic contributions. However, it was assumed that this lowest energy solution was a fair representation of the mean docking state and that variations of IE could be correlated to the protein affinity (K<sub>d</sub>) for the ligand. Following these assumptions, seven substitutions (single or double) were selected for BLG, based on the strongest decrease of interaction energy (IE) between the protein and retinol and/or PA.

*Strains and Plasmid* – All components were purchased from Invitrogen (Groeningen, The Netherlands). Competent *Escherichia coli* DH5α cells were used for cDNA cloning and the proteins were produced in the yeast strain *Pichia pastoris* X33 (Invitrogen).

The cDNA coding for BLG-A was inserted into the expression vector pGAPZαA (43), in which protein expression is controlled by the constitutive GAPDH promoter. pGAPZαA contains the α-factor secretion signal peptide from *Saccharomyces cerevisiae* and a gene conferring resistance to Zeocin™ as a selection marker.
**Site-directed mutagenesis** - The mutations were introduced by PCR on full-length, circular pGAPZαA-BLG using the following primers (from Invitrogen): primer 1A, 5'-T GCG GTG TTC AAG ATC GAT GCC TTG AAC GAG AAC-3'. Primers 1B and 1C were identical to primer 1A except that codon for residue 86 (underlined in primer 1A) was replaced by CAG (A→Q) in primer 1B or CAC (A→H) in primer 1C. Primer 2A, 5'-GG GAT CTT GGT TTT TTC TGC AAT GAT CTT CTT CTG AGC ACA-3'. Primers 2B, 2C and 2D were identical to primer 2A except that codon for residue 69 (underlined in primer 2A) was replaced by CCT (K→R) in primer 2B, GTT (K→N) in primer 2C or ATA (K→Y) in primer 2D. Primer 1A, 1B or 1C was used in combination with primer 2A, 2B, 2C or 2D according to the desired mutation(s).

The PCR products were digested with *Dpn*I, ligated overnight at 16°C with T4 DNA Ligase (New England Biolabs, Beverly, MA) and transformed by heat-shock into *E. coli*. Selection was carried out on LB-agar plates supplemented with Zeocin™ (25 µg/ml). Plasmids from positive clones were checked by sequencing (MWG Biotech, Ebersberg, Germany), linearized with *Bsp*H1 and electroporated into *P. pastoris* according to the EasySelect™ *Pichia* Expression Manual from Invitrogen. The best producing clones were selected by cultivation for 48 h in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30°C under shaking (250 rpm). Protein expression was assayed by SDS-PAGE.

**Preparation of native BLG** - BLG-A purified from cow's milk according to Mailliart and Ribadeau Dumas (44) was used as a standard in all experiments. As revealed from RP-HPLC and SDS-PAGE, the protein was more than 95% pure.

**Protein expression and purification** - A fresh colony of *P. pastoris* was grown overnight in 200 ml YPD at 30°C under strong shaking (300 rpm). This culture was used to inoculate a 3 l
fermentor Bio Console ADI 1025 from Applikon (Schiedam, the Netherlands) filled with 1.8 l of BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 0.04% biotin, 2% dextrose, 100 mM potassium phosphate buffer, pH 7.0). Protein expression was performed by cultivation during 24 h at 30°C. pH was maintained at 7.0 using 4 N NaOH. Dissolved O₂ concentration was maintained at a value over 25% by agitation (500 to 800 rpm) and aeration controlled by injection of air, and pure O₂ after 8 h of growth. Cell growth was followed by OD₆₀₀ measurement and protein expression was controlled by SDS-PAGE. After 24 h, the supernatant was collected by centrifugation (40 min, 4000 rpm).

Protein purification was carried out as previously described (45). Briefly, the supernatant was filtered, dialyzed overnight and the retentate was loaded on an anion-exchange chromatography column (Q-Sepharose XL Streamline from Pharmacia, Peapack, NJ). The protein was eluted by a 3-step NaCl gradient (0.1, 0.3 and 0.5 M). The fraction containing recombinant BLG was concentrated and purified by gel permeation chromatography (Superdex 75, Pharmacia). The purity of the eluted fractions was checked by RP-HPLC; fractions containing pure (> 95%) protein were pooled, dialyzed against deionised water and lyophilised for storage at +4°C.

**SDS-PAGE** - Electrophoresis was performed on a 15% acrylamide gel in Tris - Tricine buffer as previously described (45).

**Molar extinction coefficients calculation** - The molar extinction coefficients (ε₂₇₈) of the recombinant proteins were evaluated by applying the Beer-Lambert equation (Absorbance = ε × L × C, where “L” is the optical path length in cm and “C” is the concentration in M) on protein samples which concentration was calculated using the Micro BCA Protein Assay Kit from Pierce Biotechnology (Rockford, IL).
*Mass spectrometry* - BLG mass spectra were acquired by ESI using an ion-trap analyser (LCQ Advantage from Thermo Finnigan, San Jose, CA). The protein samples were prepared at a concentration of 5 µM in H₂O-acetonitrile (1/1, V/V) containing 0.5% (V/V) formic acid and injected by infusion at a flow-rate of 2.5 µl/min.

The spectra of BLG - ligand complexes were acquired by ESI using a Time Of Flight analyser (Q-TOF Ultima from Micromass, Manchester, U.K.). The protein samples were prepared at a concentration of 10 µM in 10 mM ammonium acetate, pH 7 and dialyzed against this buffer during 4 h. The complexes were prepared by addition of retinol or PA (in ethanol) for a [ligand]/[protein] ratio of 5 and a final ethanol concentration < 1%. The samples were injected by infusion at a flow-rate of 5 µl/min. The pressure at the interface between the ionisation chamber and the analyser was set at 4 mbars. The spectra were analysed with Protein Lynx software (Micromass).

*Circular dichroism* - The CD spectra were acquired using a CD6 dichrograph and recorded with the CDmax software (Jobin Yvon, Longjumeau, France). Protein samples were prepared at a concentration of 1.5 mg/ml in 10 mM sodium phosphate, pH 6.7 or in 10 mM glycine-HCl, pH 2. Near (250-320 nm) and far (185-260 nm)-UV spectra were measured in a 5 mm optical path length cylindrical cell and a 0.1 mm optical path length square cell, respectively. Five spectra were accumulated for each sample. The buffer spectrum was subtracted before calculation of molar ellipticity (46).

*Fluorescence spectroscopy* - Fluorescence spectroscopy experiments were carried out at 20°C using an Aminco SLM 4800C spectrofluorimeter (Thermo Spectronic, Rochester, NY) in the ratio mode. The proteins were prepared at a concentration of 10 µM in 50 mM sodium
phosphate, pH 6.7 and incubated for 15 min at 20°C under agitation before any measurement.

The excitation wavelength was set at 290 nm and the slits were at 4 nm (excitation) and 8 nm (emission). The ligands were all-*trans*-retinol and sodium palmitate from Sigma (St Louis, MO). For titrations, ligands were solubilized in ethanol at a concentration of 0.5 mM. The proteins were titrated by addition of small increments (2-4 µl) of ligand; the ethanol final concentration never exceeded 4%. For each measurement, the solution was equilibrated 3 min under agitation before measuring the fluorescence emission intensity at 330 nm (average of 15 values). Blank experiments were performed with N-acetyl-tryptophanamide prepared at the same OD₂₉₀ as the protein (retinol experiments) or BLG titrated with ethanol (PA experiments).

*Binding parameters calculation* - The dissociation constant (K_d) and the number of binding sites per protein monomer (N) were calculated using a non-linear Newton-Gauss function previously successfully used for the analysis of the interactions between hydrophobic ligands and Lipid-Binding Proteins (47):

$$ \alpha^2 - (1 + R_i/N + K_d/P_t) \alpha + R_i/N = 0 \quad (\text{Eq. 1}) $$

$$ \text{with } \alpha = (F - F_0) / (F_{\text{max}} - F_0) $$

R_i is the [ligand]/[protein] ratio for each experimental point. P_t is the total protein concentration. F_0, F and Fmax are the values of protein fluorescence intensity in the absence of ligand, at each experimental point and at the saturation, respectively. Eq. 1 was resolved for each experimental point using Grafit software from Erithacus (Horley, U.K.).
RESULTS

*Determination of native BLG - ligand structures* - The mutations were chosen by molecular modeling simulations, using the crystal structures of BLG - retinol (PDB coordinates 1GX8) and BLG - PA (PDB 1B0O) complexes as templates. However, because of molecular mobility, other structures than those selected during the crystallization process may exist in solution. Therefore, molecular modeling simulations were carried out in order to define these alternate structures by orientating the BLG - ligand complexes towards new local energy minima. Calculations were performed with neither the water molecules nor the periodic packing conditions of the crystal. The effects of pH and of the presence of water were approximated by systematic protonation of acidic amino-acid residues and of palmitate ion and by performing all calculation experiments at pH 7.0, as previously described (48). For each BLG - ligand complex, an alternate structure corresponding to the best local energy minimum could be defined (see Experimental Methods section).

In the new structures, both ligands and several amino acid residues located at the entry of the hydrophobic calyx are repositioned (Fig. 2). In our model for BLG - retinol complex, the ligand global position is similar to that determined in the crystallographic study, with its OH group oriented towards residues E62 and K69 (Fig. 2, A and B). However, the crystal structure describes a unique H-bond with residue E62, whereas in our model the retinol OH group is bonded to both OE2 of E62 and NZ of K69. Our model also describes a H-bond between residues K69 and E62 that does not appear in the crystal structure.

The model for BLG - PA complex derived from energy minimization is significantly different from the crystal structure (Fig. 2, C and D). In the latter, the ligand carboxylate group is only involved in two H-bonds with residues K60 and K69. In our model, the carboxylate group of PA is strongly repositioned with a rotation of about 90° compared to its
crystallographic position, yielding significant changes in the H-bonds network. The O1 interacts with residue E62, which is itself bonded to residue K69 while the O2 of the ligand is bonded to both ND of N88 and to the oxygen atom in the L87-N88 peptide bond. Residues L87 and N88 belong to the protein CD loop (Fig. 1), whose interaction with PA was not observed in the crystallographic studies (26, 27). It appears from molecular modeling simulations and energy refinement as applied here, that PA binding to residues located at both sides of the calyx entry is possible and leads to a stronger interaction with BLG compared to the crystal structure.

However, the model defined by energy minimization leads to the "statistically most probable structure" which does not necessarily reflect the dynamic interchanges between a variety of conformations that might exist in solution. The calculated structures described above cannot be considered as the only possible conformation for the complex, or indeed any 'better' than that observed in the crystal, mainly because they do not take into account the micro-arrangements of water molecules at the protein-ligand interface. Nevertheless, because of the energy minimization procedure adopted, our models can be expected to pinpoint one of the more energetically favourable conformations possible for the complexes.

*Choice of the mutations* – Visual examination of the native structures revealed that residues K69 and A86, located in the vicinity of the ligands' polar groups, were good candidates for mutation (Fig. 2, A and C):

- K69 interacts with retinol but not with PA. Modification of its side-chain might allow an interaction with PA and/or improve its interaction with retinol;

- A86 is located on the CD loop, its side-chain pointing towards the entry of the calyx. However, the side-chain is far from the ligand and contains no functional group. A
substitution at this position that introduces a polar or ionisable side-chain might allow new
bonds between BLG and retinol and/or PA.

Several single and double substitutions at positions 69 and 86 were tested by molecular modeling. For each of them, the structure was refined by energy minimization as before and the protein-ligand interaction energy was calculated. This procedure identified seven mutations that could be expected to increase BLG affinity for retinol and/or PA. They were four single substitutions (K69R, K69N, K69Y and A86Q) and three double substitutions (K69R-A86Q, K69N-A86Q and K69Y-A86H).

*Production and purification of BLG mutants* - Wild-Type (WT) and the seven mutants of BLG were expressed in the yeast *P. pastoris* and the proteins were purified by ion exchange and gel permeation chromatography. All proteins were purified successfully, except for the K69Y mutant, which aggregated or unfolded during purification. This mutant was not studied further. Over-loaded SDS-PAGE (Fig. 3) and RP-HPLC (not shown) indicated that recombinant proteins purity was > 95%. All mutants retained their dimeric form at neutral pH, as observed by gel permeation HPLC (data not shown). Mass spectrometry confirmed the successful introduction of the mutations and the absence of post-translational modifications (data not shown). The yield of pure protein was between 40 and 200 mg/l of supernatant. The molar extinction coefficient at 278 nm (ε<sub>278</sub>) of all proteins was found equal to that of native protein (17.600 M<sup>-1</sup>, Ref. 5) except for the K69Y-A86H mutant (21.000 M<sup>-1</sup>).

*CD spectra at neutral and acidic pH* – The CD spectra of the BLG mutants at pH 6.7 are similar to that of native protein in both the far- and near-UV regions (Fig. 4, A and B). This result indicates that all mutants are folded with native-like secondary and tertiary structures. However, the CD spectra for A86Q and K69Y-A86H mutants show a lower ellipticity in the
185-195 nm and the 260-285 nm regions, respectively. This cannot be attributed to protein unfolding, which leads to a decrease of ellipticity over the whole spectrum (49).

BLG is stable at acidic pH. When the pH is < 3, the monomer ↔ dimer equilibrium is shifted to almost totally to the monomer form, which retains a β-barrel fold but with increased compactness and rigidity (50, 51). This unique and rigid form is easier to characterize than the flexible, dynamic structure at neutral pH (52). CD spectra of BLG mutants at pH 2.0 are all superimposable with that of the native protein in both far- and near-UV regions (Fig. 4, C and D). These results confirm that each of the mutants is correctly folded, and that the differences observed in the CD spectra at neutral pH are mainly due to local modifications of the protein structural dynamics. Unsurprisingly, these changes are observed in the case of the A86Q and K69Y-A86H substitutions, which are the least conservative amino acid changes to BLG.

BLG titration with retinol and PA - Native BLG-A (purified from bovine milk) and recombinant WT-BLG were titrated with retinol and PA. The binding was followed by measuring the change in protein fluorescence emission at 330 nm (Fig. 5) and the binding parameters were calculated from a non-linear, mathematical model (see Experimental Methods section). The K_d values for retinol and PA binding to WT-BLG were 2.64 and 0.44 µM, respectively (Table 1A). These values are equivalent to those obtained with native BLG purified from milk. The K_d for PA is similar to that determined in previous studies (from 0.1 to 0.6 µM, Ref 10 and 53), whereas the K_d for retinol is higher than those previously calculated (between 36 nM and 0.6 µM, Ref 12, 27, 32, 54). However, in all previous studies the binding parameters were calculated with a linear model, which systematically leads to an under-estimation of K_d in the case of retinoid binding to BLG (55).

The number of theoretical binding sites per protein monomer (N) was 0.5 for retinol and 0.7 for PA (Table 1A). These values contrast with those calculated in the previous
experiments quoted above, where N was between 0.8 and 1. Such discrepancy may result
from the use of a non-linear mathematical model in the present study (see Discussion, below)
but is most probably arises from the inaccuracy of determination of the value of Fmax.
Nevertheless, mass spectrometry experiments on BLG complexes with retinol and PA
demonstrate the existence of one strong binding site for both ligands per protein monomer
(Fig. 6). This result is similar to that obtained with *cis*-parinaric acid (11) and confirms a 1:1
stoichiometry for retinol and PA to the BLG monomer, despite the calculated N values.

Table 1B shows the binding parameters obtained from titrations of BLG mutants with
retinol and PA. The N values are all close to those calculated for both native and wild-type
BLG, indicating that the relative binding stoichiometry was unmodified by the mutations. The
affinity of the K69N and A86Q mutants for retinol was similar to that of WT-BLG. In all
other cases, the protein affinity for both ligands was increased, from 2- to 5.5-fold in the case
of retinol and from 2- to 10-fold in the case of PA. The largest increase in affinity was for the
K69R-A86Q substitution with retinol, and for the K69N-A86Q substitution with PA. These
results are, to the best of our knowledge, the first report of an increase in BLG affinity for its
ligands following site-directed mutagenesis experiments.

*Correlation between experimental results and molecular modeling* - Table 1 and Figure 7 show the correspondence between K<sub>d</sub> values calculated from fluorescence experiments and the interaction energy (IE) obtained from molecular modeling simulations, for all proteins. The equation IE = f (Log K<sub>d</sub>) is a linear relation for 4 mutants out of the 6 in the case of retinol (Fig. 7A) and for 5 mutants out of 6 in the case of PA (Fig. 7B). The linear correlation is not valid for K69N for either ligand or for K69Y-A86H in the case of retinol. However, the correlation observed in most cases demonstrates that the IE calculated from molecular
modeling simulations provides a reasonable estimate of the effect of BLG substitutions on its
affinity for retinol and PA.

DISCUSSION

*Molecular modeling efficiency* - Site-directed mutagenesis of BLG has led to an increase of its affinity for retinol and for PA. The mutagenesis strategy was based on molecular modeling simulations and, in most cases, the experimental results correlated with the effects predicted by the simulations. Such a correlation demonstrates that molecular modeling can be an efficient support for the site-directed mutagenesis of BLG. However, this correlation requires energy minimization of the crystal structures, taking into account both ligand and protein mobility.

The interpretation of the experimental results obtained by molecular modeling
simulations is limited by i) the incomplete evaluation of ionic interactions and ii) the absence
of specific water molecules. As crystallographic refinement generally only locates a few water
molecules around the protein surface, the picture provided is incomplete. Similarly, molecular
modeling experiments like those performed here, are based upon an approximate structure, in
which the effects of water molecules can only be mimicked by partial protonation of the
acidic amino acid residues (48). This imperfection probably results in the discrepancies
observed in Fig. 7. In the case of K69Y-A86H mutant complexed with retinol, our model
introduces a distortion of the residue H86 side-chain, which is oriented towards the carbonyl
oxygen of the H86-L87 peptide bond (not shown). Although such bending of H86 in aqueous
solution is improbable, it was systematically created during the cycles of energy
minimization. Thus, we were unable to build a reliable model for this complex, which may
explain the absence of correlation. In the case of K69N, the absence of correlation between simulation and experiment with both ligands remains unexplained.

*Thermodynamic parameters calculation* - Traditionally, the thermodynamic parameters for ligand binding to BLG ($K_d$ and N) are calculated using the linear mathematical model first proposed by Cogan and coworkers (56). This model requires calculation of the free ligand concentration ($L_f$), which may be directly obtained when the binding is followed by ligand fluorescence. When the protein fluorescence is followed, $L_f$ cannot be directly measured and can only be estimated using the relation:

$$L_f = L_t - L_b \qquad (\text{Eq. 2})$$

where $L_t$ and $L_b$ are the total and bound ligand concentrations, respectively, and $L_b$ is calculated from experimental data.

However, Eq. 2 is not valid when the ligand is insoluble or only sparingly soluble in aqueous buffers: a large part remains in an aggregated or micellar form and the concentration of free, available ligand is systematically lower than the estimated value. Ligand insolubility is thus a problem when titrating BLG with retinol or PA: $6.10^{-8}$ M and $10^{-10}$ M, respectively (57, 58). Thus, in order to avoid errors in calculated quantities arising from the over-estimation of $L_f$, it is advisable to use equations in which $L_f$ does not appear (59-61).

Another important drawback of the fit proposed by Cogan's group is the necessity of estimating the maximum (or minimum) fluorescence intensity value. This subjectivity has been shown to be responsible for $K_d$ under-estimation (55). A possible aggregation of BLG during titration experiments has also been pointed out, which would also lead to inaccurate thermodynamic parameters (28). Consequently, we chose to calculate the binding parameters as described in the Experimental Methods section. This equation contains only N, $K_d$ and $F_{max}$
as parameters and allows the calculation of relative values for each of them with no need for
estimation of Lf nor subjective definition of Fmax (47).

The N values obtained in our study are lower than those calculated previously, i. e. 0.5 and 0.7 instead of 0.9-1 and 0.8 for retinol and PA, respectively (10, 12, 27, 53-55). However, the latter values were calculated using the linear mathematical model, which fitted only a few of the experimental points (28, 55). The non-linear equation used in this study evaluates the binding parameters from the whole experimental data set. By using a subset of the experimental points, it was possible to obtain N values much closer to 1, although there is no real justification for the selection (not shown).

We conclude that the relative values calculated in our study are representative of the
complexity of low-soluble ligands binding to BLG and are not in disagreement with the
theoretical 1:1 stoichiometry. Obtaining absolute values would require a full description of all
equilibriums occurring in solution, which is presently impossible. However, the non-linear
model used in our study allows calculation of reproducible values from the whole set of
experimental data, and therefore constitutes a useful and reliable tool for the analysis of ligand
binding to BLG independently of the underestimation of binding stoichiometries.

*Structural analysis of the BLG - retinol complex* - The mutations led neither to a significant repositioning of retinol inside the calyx nor to significant movement of its polar group. Whatever the mutation, the ligand is oriented towards the residue at position 69 (Fig. 8). In the case of the K69N substitution, the ligand is slightly repositioned in order to maintain a H-bond with N69. However, this repositioning has no effect on BLG affinity for retinol (Table 1). The A86Q and A86H substitutions do not induce a rotation of retinol OH group towards the glutaminyl or histidyl residues, due to the low flexibility of retinol caused by its polyene chain. The low mobility of retinol may also be the main reason why energy refinement of the
BLG - retinol crystal structure led to slight repositioning of the ligand inside the calyx (Fig. 2, A and B). Thus, the position observed after energy minimization, which is essentially that observed in the crystal structure, is most probably the only one possible.

Only the two mutants containing a K69R substitution induce a significant increase of BLG affinity for retinol (Table 1). A possible explanation is the longer side-chain of the arginyl residue, what allows deeper burying of retinol inside the calyx and may lead to stronger hydrophobic interactions with the protein. The increase of affinity may also be due to the presence of a guanidinium group in the arginyl residue side-chain: at neutral pH this group is protonated and highly stabilized through electronic resonance. An electron from the retinol oxygen atom can participate in the resonance process, leading to a strong interaction between R69 and retinol. Such an interaction is expected to be more stable than the unique H-bond between the retinol OH group and the amino group of the lysyl residue.

*Structural analysis of the BLG - PA complex* - Depending on the mutation, PA can occupy three different positions (Fig. 9). In position 1, PA is bonded to N88, E62 and L87 (WT) or Q86 (A86Q and K69N-A86Q mutants). In position 2, the ligand is away from N88 and is bonded to E62, L87 (K69R and K69N mutants), Q86 (K69R-A86Q mutant) or R69 (K69R mutant). In the case of the K69Y-A86H mutant, PA is oriented in a third position and is bonded to P38, E62 and H86.

Thus, the flexibility of PA allows it to adapt to the different environments at the entrance to the calyx, depending on the amino acid residues available in its neighbourhood. This higher adaptability, compared to that of retinol, arises from the fully saturated aliphatic chain and the presence of two oxygen atoms in its carboxylate and explains why energy minimization of the BLG - PA crystal structure led to an important repositioning of the ligand inside the calyx. It also explains why the substitutions have a stronger effect on BLG affinity
for PA than for retinol: the greater mobility of the PA allows it to adapt more specifically to
the modified geometry of the binding site. It is worth noting that the crystal structures of the
C₁₄ – C₁₈ series of aliphatic monocarboxylic acids show a significant variation in the positions
of polar head groups (to be published).

*Affinity and H-bond network geometry* - The strength of the H-bonded network, which is in part responsible for the ligand affinity, is dependent both upon the orientation and length between donor and acceptor (62, 63, 64, 65). In an extended network, the preferred geometry is tetrahedral and it is no surprise that that is what we observe in the case of the BLG mutant interactions with PA:

- K69R, K69N and K69R-A86Q mutations induce a small increase of BLG affinity for PA (Table 1B). In these three mutants, as well as in WT-BLG, the putative H-bond networks are created with sub-optimal geometries, especially those involving residues L87 or A/Q86 (Fig. 2 C and Fig. 9B, C and E).

- The A86Q substitution induces a 4-fold increase of affinity, and the H-bond network is close to the most favourable geometry (Fig. 9D).

- The K69Y-A86H substitution leads to a 6-fold increase of BLG affinity. In this mutant the ligand is H-bonded to E62 and on the opposite side by residues P38 and H86 (Fig. 9G). The orientations of the two latter bonds are favourable.

- In the case of K69N-A86Q, BLG affinity for PA is 10-fold increased, which is the highest increase observed in our experiments. Here, the modeled structure shows a typical network involving residues E62, N88 and Q86 (Fig. 9F). Moreover, no H-bond is created between residues E62 and N69, which is a unique event within our structures for BLG - retinol and BLG - PA complexes. It can be expected that the release of E62 from its bonding to residue at position 69 reinforces its interaction with the ligand. This reinforcement together with the
favourable H-bond network provides a possible explanation for the significant increase of affinity observed with this mutant.

CONCLUSION

We have produced and purified BLG mutants designed to increase BLG affinity for retinol and PA. The mutants were chosen on the basis of molecular modelling simulations, which appear to be an efficient tool for such studies. Despite imperfections from the approximations used in the force field calculations, notably the absence of explicit water molecules, molecular modelling was able to indicate suitable mutations, which have been shown experimentally to have improved binding properties.

The mutations led to an increase of affinity for both ligands (5.5-fold for retinol and 10-fold for PA) and in the majority of cases the experimental results correlated with the simulations. These experiments showed us several important parameters contributing to the strength of BLG - ligand interaction:

- the flexibility or mobility of the ligand

- the number and nature of ligand functional groups available for interaction with the protein (retinol vs. PA);

- the involvement of the loops located at the entry of the calyx (CD vs. CD + EF loops);

- the capacity of the amino acid residue side-chains on these loops to create strong interactions with the ligand polar group and/or to allow burying of the ligand inside the pocket (arginyl vs. lysyl);

- the geometry of the H-bond network formed between the ligand polar group and the protein.

The results presented in this study are of significant interest for understanding the mechanisms that drive protein - ligand interaction, and more specifically in the case of
sparingly soluble ligands like retinoids and fatty acids binding to bovine BLG. Other site-
directed mutagenesis experiments on BLG can be considered, based on the present results, in
order to increase its affinity for ligands of interest. Nevertheless, the results obtained here are
still insufficient for pharmaceutical applications and must be completed by more powerful
strategies, such as semi-random or random mutagenesis and techniques of molecular
evolution.

ACKNOWLEDGEMENTS

The authors thank Hélène Rogniaux and François Métro (INRA, BIA) for the mass
spectrometry experiments, and Didier Marion for discussions and advice concerning the non-
linear analysis of the interactions of hydrophobic ligands with the mutant BLGs.

REFERENCES

1. Papiz, M.Z., Sawyer, L., Eliopoulos, E.E., North, A.C., Findlay, J.B., Sivaprasadarao, R., Jones, T.A., Newcomer, M.E., and Kraulis, P.J. (1986) The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein, *Nature* **324**, 383-385

2. Åkerstrom, B., Flower, D. R., and Salier, J.-P. (2000) Lipocalins: unity in diversity, *Biochim. Biophys. Acta* **1482**, 1-8

3. Sakurai, K., and Goto, Y. (2002) Manipulating monomer-dimer equilibrium of bovine β-lactoglobulin by amino acid substitution, *J. Biol. Chem.* **277**(28), 25735-25740

4. Fugate, R. D., and Song, P.S. (1980) Spectroscopic characterization of β-lactoglobulin – retinol complex, *Biochim. Biophys. Acta* **625**, 28-42
5. Dufour, E., and Haertlé, T. (1991) Binding of retinoids and β-carotene to β-lactoglobulin. Influence of protein modifications, *Biochim. Biophys. Acta* **1079**, 316-320

6. Wang, Q., Allen, J. C., and Swaisgood, H. E. (1999) Binding of lipophilic nutrients to β-lactoglobulin prepared by bioselective absorption, *J. Dairy Sci.* **82**, 257-264

7. Zsila, F., Bikadi, Z., and Simonyi, M. (2002) Retinoic acid binding properties of the lipocalin member β-lactoglobulin studied by circular dichroism, electronic absorption spectroscopy and molecular modelling methods, *Biochem. Pharmacol.* **64**, 1651-1660

8. Spector, A. A., and Fletcher, J. E. (1969) Binding of long-chain fatty acids to β-lactoglobulin, *Lipids* **5**(4), 403-411

9. Puyol, P., Perez, M. D., Ena, J. M., and Calvo, M. (1991) Interaction of bovine β-lactoglobulin and other bovine and human whey proteins with retinol and palmitic acid, *Agric. Biol. Chem.* **55**(10), 2515-2520

10. Frapin, D., Dufour, E., and Haertlé, T. (1993) Probing the fatty-acid binding site of β-lactoglobulins, *J. Protein Chem.* **12**(4), 443-449

11. Diaz de Villegas, M. C., Oria, R., Sala, F. J., and Calvo, M. (1987) Lipid binding by β-lactoglobulin of cow milk, *Milchwissenschaft* **42**(6), 357-358

12. Wang, Q., Allen, J. C., and Swaisgood, H. E. (1997) Binding of vitamin D and cholesterol to β-lactoglobulin, *J. Dairy Sci.* **80**, 1054-1059

13. Guichard, E., and Langourieux, S. (2000) Interactions between β-lactoglobulin and flavor compounds, *Food Chem.* **71**, 301-308

14. van Ruth, S. M., and Villeneuve, E. (2002) Influence of β-lactoglobulin, pH and presence of other aroma compounds on the air/liquid partition coefficients of 20 aroma compounds varying in functional group and chain length, *Food Chem.* **79**, 157-164

15. Sawyer, L., Brownlow, S., Polikarpov, I., and Wu, S.-Y. (1998) β-lactoglobulin: structural studies, biological clues, *Int. Dairy J.* **8**, 65-72
16. Hemley, R., Kohler, B. E., and Siviski, P. (1979) Absorption spectra for the complexes formed from vitamin A and β-lactoglobulin, *Biophys. J.* 28, 447-455

17. O'Neill, T., and Kinsella, J. E. (1987) Binding of alkanone flavors to β-lactoglobulin: effects of conformational and chemical modifications, *J. Agric. Food Chem.* 35, 770-774

18. Dufour, E., and Haertlé, T. (1990) Binding affinities of β-ionone and related flavor compounds to β-lactoglobulin: effects of chemical modifications, *J. Agric. Food Chem.* 38, 1691-1695

19. Busti, P., Scarpeci, S., Gatti, C. A., and Delorenzi, N. J. (1999) Interaction of alkylsulfonate ligands with β-lactoglobulin AB from bovine milk, *J. Agric. Food Chem.* 47, 3628-3631

20. Zsila, F. (2003) A new ligand for an old lipocalin: induced circular dichroism spectra reveal binding of bilirubin to bovine β-lactoglobulin, *FEBS Lett.* 539, 85-90

21. Collini, M., D'Alfonso, L., Molinari, H., Ragona, L., Catalano, M., and Baldini, G. (2003) Competitive binding of fatty acids and the fluorescent probe 1-anilino-8-naphthalenesulfonate to bovine β-lactoglobulin, *Protein Sci.* 12, 1596-1603

22. Qin, B. Y., Creamer, L. K., Baker, E. N., and Jameson, G. B. (1998) 12-bromododecanoic acid binds inside the calyx of bovine β-lactoglobulin, *FEBS Lett.* 438, 272-278

23. Wu, S.-Y., Perez, M. D., Puyol, P., and Sawyer, L. (1999) β-lactoglobulin binds palmitate within its central cavity, *J. Biol. Chem.* 274(1), 170-174

24. Kontopidis, G., Holt, C., and Sawyer, L. (2002) The ligand-binding site of bovine β-lactoglobulin: evidence for a function?, *J. Mol. Biol.* 318, 1043-1055

25. Chen, S. X., Hardin, C. C., and Swaisgood, H. E. (1993) Purification and characterization of β-structural domains of β-lactoglobulin liberated by limited proteolysis, *J. Protein Chem.* 12(5), 613-625
26. Imre, T., Zsila, F., and Szabo, P. T. (2003) Electrospray mass spectrometric investigation of the binding of cis-parinaric acid to bovine β-lactoglobulin and study of the ligand-binding site of the protein using limited proteolysis, *Rapid Commun. Mass Spectrom.* **17**(22), 2464-2470

27. Cho, Y., Batt, C.A. and Sawyer, L. (1994) Probing the retinol-binding site of bovine β-lactoglobulin, *J. Biol. Chem.* **269**, 11102-11107

28. Wang, Q., Allen, J. C., and Swaisgood, H. E. (1998) Protein concentration dependence of palmitate binding to β-lactoglobulin, *J. Dairy Sci.* **81**, 76-81

29. Collini, M., D'Alfonso, L., and Baldini, G. (2000) New insight on β-lactoglobulin binding sites by 1-anilino-8-naphthalenesulfonate fluorescence decay, *Protein Sci.* **9**, 1968-1974

30. Patel, R. C., Lange, D., McConathy, W. J., Patel, Y. C., and Patel, S. (1997) Probing the structure of the ligand cavity of lipocalins by fluorescence spectroscopy, *Protein Eng.* **10**(6), 621-625

31. Lübke, M., Guichard, E., Tromelin, A., and Le Quéré, J.-L. (2002) Nuclear magnetic resonance spectroscopy of β-lactoglobulin interactions with two flavor compounds, γ-decalactone and β-ionone, *J. Agric. Food Chem.* **50**, 7094-7099

32. Narayan, M., and Berliner, L. J. (1997) Fatty acids and retinoids bind independently and simultaneously to β-lactoglobulin, *Biochemistry* **36**, 1906-1911

33. De Wolf, F. A., and Brett, G. M. (2000) Ligand-binding proteins: their potential for application in systems for controlled delivery and uptake of ligands, *Pharmacol. Rev.* **52**(2), 207-236

34. Verhoeyen, M. E., van der Loft, C. P. E., Beggs, T. S., and Davis, P. J. (1995) Antibody fragments for controlled delivery of therapeutic agents, *Biochem. Soc. Trans.* **23**, 1067-1073

35. Carcenac, M., Dorvillius, M., Garambois, V., Glaussel, F., Larroque, C., Langlois, R., Hynes, N. E., van Lier, J. E., and Pelegrin, A. (2001) Internalization enhances photo-induced
cytotoxicity of monoclonal antibody – phthalocyanine conjugates, *British J. Cancer* 85(11), 1787-1793

36. Markussen, J., Havelund, S., Kurtzhals, P., Andersen, A. S., Halstrom, J., Hasselager, E., Larsen, U. D., Ribel, U., Schaffer, L., Vad, K., and Jonassen, I. (1996) Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs, *Diabetologia* 39, 281-288

37. Fillebeen, C., Descamps, L., Dehouck, M. P., Fenart, L., Benaissa, M., Spik, G., Cecchelli, R., and Pierce, A. (1999) Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier, *J. Biol. Chem.* 274, 7011-7017

38. Schechter, B., Chen, L., Arnon, R., and Wilchek, M. (1999) Organ selective delivery using a tissue-directed streptavidin-biotin system: targeting 5-fluorouridine via TNP-streptavidin, *J. Drug Target* 6(5), 337-348

39. McAlpine, A. S., and Sawyer, L. (1990) β-lactoglobulin: a protein drug carrier?, *Biochem. Soc. Trans.* 18, 879

40. Sawyer, L. (1991) The role and structure of β-lactoglobulin, in *Protein engineering. Proceedings of an AFRC conference on protein engineering in the agricultural, and food industry* (Goodenough, P., Ed.), CPL Press, Newbury, U.-K.

41. Batt, C. A., Brady, J., and Sawyer, L. (1994) Design improvements of β-lactoglobulin, *Trends Food Sci. Tech.* 5, 261-265

42. Skerra, A. (2000) Lipocalins as a scaffold, *Biochim. Biophys. Acta* 1482, 337-350

43. Wilson, C., Quarrie, L., Allan, G. J., Flint, D. J., Sawyer, L., and Holt, C. (1999) Expression of recombinant wild-type and mutant β-lactoglobulins in the yeast *Pichia pastoris*, *Int. J. Food Sci. Tech.* 34, 445-450
44. Mailliart, P. and Ribadeau Dumas, B. (1988) Preparation of β-lactoglobulin and β-lactoglobulin-free proteins from whey retentate by NaCl salting out at low pH, *J. Food Sci.* **53**, 743-752

45. Jayat, D., Gaudin, J.-C., Chobert, J.-M., Burova, T., Holt, C., McNae, I., Sawyer, L., and Haertlé, T. (2004) A recombinant C121S mutant of bovine β-lactoglobulin is more susceptible to peptic digestion and to denaturation by reducing agents and heating, *Biochemistry* **43**(20), 6312-6321

46. Kelly, S. M., and Price, N. C. (2000) The use of circular dichroism in the investigation of protein structure and function, *Curr. Prot. Peptide Sci.* **1**(4), 349-384

47. Dubreil, L., Compoint, J.-P., and Marion, D. (1997) Interaction of puroindulines with wheat flour polar lipids determines their foaming properties, *J. Agric. Food Chem.* **45**, 108-116

48. Woolf, T. B. (1998) Simulations of fatty acid-binding proteins suggest sites important for function. I. Stearic acid, *Biophys. J.* **74**, 681-693

49. Manderson, G.A., Hardman, M.J. and Creamer, L.K. (1999) Effect of heat treatment on bovine β-lactoglobulin A, B, and C explored using thiol availability and fluorescence, *J. Agric. Food Chem.* **47**, 3617-3627

50. Uhrinova, S., Smith, M., Jameson, G. B., Uhrin, D., Sawyer, L., and Barlow, P. N. (2000) Structural changes accompanying pH-induced dissociation of the β-lactoglobulin dimer, *Biochemistry* **39**, 3565-3574

51. Fogolari, F., Ragona, L., Zetta, L., Romagnoli, S., De Kruif, K. G., and Molinari, H. (1998) Monomeric bovine β-lactoglobulin adopts a β-barrel fold at pH 2, *FEBS Lett.* **436**, 149-154

52. Burova, T.V., Choiset, Y., Tran, V., and Haertlé, T. (1998) Role of free Cys121 in stabilization of bovine β-lactoglobulin B, *Prot. Eng.* **11**, 1065-1073
53. Narayan, M. and Berliner, L.J. (1998) Mapping fatty acid binding to β-lactoglobulin, *Protein Sci.* 7, 150-157

54. Dufour, E., Genot, C., and Haertlé, T. (1994) β-lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy, *Biochim. Biophys. Acta* 1205, 105-112

55. Muresan, S., van der Bent, A., and de Wolf F. A. (2001) Interaction of β-lactoglobulin with small hydrophobic ligands as monitored by fluorimetry and equilibrium dialysis: nonlinear quenching effects related to protein – protein association, *J. Agric. Food Chem.* 49, 2609-2618

56. Cogan, U., Kopelman, M., Mokady, S., and Shinitzky, M. (1976) Binding affinities of retinol and related compounds to retinol binding proteins, *Eur. J. Biochem.* 65, 71-78

57. Szuts, E. Z., and Harosi, F. I. (1991) Solubility of retinoids in water, *Arch. Biochem. Biophys.* 287(2), 297-304

58. Vorum, H., and Brodersen, R. (1994) Adsorption and physical state of medium and long-chain fatty acids in neutral aqueous buffer solutions, *Chem. Phys. Lipids* 74, 43-48

59. Brodersen, R., Vorum, H., Skriver, E., and Pedersen, A. O. (1989) Serum albumin binding of palmitate and stearate – multiple binding theory for insoluble ligands, *Eur. J. Biochem.* 182, 19-25

60. Brodersen, R., Vorum, H., and Andersen, S. (1994) Measurement of palmitate availability in serum samples: method and utility, *Chem. Phys. Lipids* 71, 13-20

61. Richieri, G. V., Ogata, R. T., and Kleinfeld, A. M. (1994) Equilibrium constants for the binding of fatty acids with fatty acids-binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB, *J. Biol. Chem.* 269(39), 23918-23930

62. Saebo, T. O., and Talberg, H.-J. (1976) The effect of hydrogen bonding on structural parameters : VI. The angular dependence of the hydrogen bond strength-AN *ab initio*. Study
of the formamide-water and the formamide-methane complexes, *Adv. Molecular Relaxation Processes* 8(1), 43-48

63. Bako, I., Schubert, G., Megyes, T., Palinkas, G., Swan, G. I., Dore, J., and Bellisent-Funel M.-C. (2004) Structural investigation of liquid formic acid by neutron diffraction. II: Isotopic substitution for DCOO[H/D], *Chem. Phys.* 306(1-3), 241-251

64. Hartnig, C., Witschel, W., Spohr, E., Gallo, P., Ricci M. A., and Rovere, M. (2000) Modifications of the hydrogen bond network of liquid water in a cylindrical SiO₂ pore, *J. Mol. Liq.* 85(1-2), 127-137

65. Li, J.-C., Ross, D. K., Hayes, M. H. B., Sherman, W. F., and Adams, M. (1994) *Hydrogen Bond Networks* (Bellessent-Funel, M.-C., and Dore, J. C., Eds), Kluwer Academic Publishers, Dordrecht, The Netherlands
<table><caption>**Table 1.** Binding parameters and interaction energies calculated for non-mutated (A) and mutated (B) BLG. IE = Interaction Energy.</caption><thead><tr><th rowspan="2"></th><th colspan="3">RETINOL</th><th colspan="3">PALMITIC ACID</th></tr><tr><th>N</th><th>K<sub>d</sub>(10<sup>-6</sup> M)</th><th>IE (kcal/mol)</th><th>N</th><th>K<sub>d</sub> (10<sup>-6</sup> M)</th><th>IE (kcal/mol)</th></tr></thead><tbody><tr><td>BLG-A</td><td>0.56 ± 0.09</td><td>2.42 ± 0.21</td><td>-48.2</td><td>0.71 ± 0.06</td><td>0.42 ± 0.10</td><td>-57.8</td></tr><tr><td>WT</td><td>0.47 ± 0.06</td><td>2.64 ± 0.20</td><td>-48.2</td><td>0.70 ± 0.07</td><td>0.44 ± 0.10</td><td>-57.8</td></tr></tbody></table><table><thead><tr><th rowspan="2"></th><th colspan="3">B</th><th colspan="3"></th></tr><tr><th>N</th><th>K<sub>d</sub> (10<sup>-6</sup> M)</th><th>IE (kcal/mol)</th><th>N</th><th>K<sub>d</sub> (10<sup>-6</sup> M)</th><th>IE (kcal/mol)</th></tr></thead><tbody><tr><td>K69R</td><td>0.59 ± 0.05</td><td>0.68 ± 0.11</td><td>-53.7</td><td>0.74 ± 0.13</td><td>0.12 ± 0.03</td><td>-62.2</td></tr><tr><td>K69N</td><td>0.51 ± 0.05</td><td>2.48 ± 0.28</td><td>-51.5</td><td>0.59 ± 0.02</td><td>0.20 ± 0.08</td><td>-57.2</td></tr><tr><td>A86Q</td><td>0.42 ± 0.07</td><td>2.20 ± 0.29</td><td>-49.7</td><td>0.68 ± 0.11</td><td>0.10 ± 0.02</td><td>-62.9</td></tr><tr><td>K69R-A86Q</td><td>0.58 ± 0.07</td><td>0.46 ± 0.17</td><td>-55.3</td><td>0.76 ± 0.03</td><td>0.10 ± 0.03</td><td>-62.7</td></tr><tr><td>K69N-A86Q</td><td>0.59 ± 0.06</td><td>1.20 ± 0.22</td><td>-52.3</td><td>0.72 ± 0.05</td><td>0.05 ± 0.02</td><td>-65.0</td></tr><tr><td>K69Y-A86H</td><td>0.57 ± 0.04</td><td>1.16 ± 0.20</td><td>-48.6</td><td>0.74 ± 0.03</td><td>0.07 ± 0.03</td><td>-63.9</td></tr></tbody></table>
FIGURE LEGENDS

FIGURE 1: BLG structure overview, complexed with retinol (modified from PDB 1GX8). β-strands (labeled A-I) are colored in yellow; the main α-helix is colored in red; the loops are colored in orange. The side-chains of residues K60, E62, K69, A86, L87 and N88 are shown. Retinol is shown as a ball-and-stick representation and colored in mauve. Oxygen and nitrogen atoms are colored in red and in blue, respectively. The Figure was produced by MOLSCRIPT (reference to whatever program was used)

FIGURE 2: Structures of BLG - ligand complexes derived from energy minimization and crystallographic data. A: BLG - retinol from molecular modeling; B: BLG - retinol from crystallography (PDB 1GX8); C: BLG - PA from molecular modeling; D: BLG - PA from crystallography (PDB 1B0O). Atoms are colored as in Figure 1. White sticks represent the H-bonds. For clarity, hydrogen atoms are not represented.

FIGURE 3: SDS-PAGE (15% acrylamide) of BLG mutants. S = molecular mass standards.
Lane 1: WT-BLG; lane 2: K69R; lane 3: K69N; lane 4: A86Q; lane 5: K69R-A86Q; lane 6: K69N-A86Q; lane 7: K69Y-A86H. The main bands for WT-BLG and the mutants are identical to that of native BLG (see Fig. 3, ref. 45).

FIGURE 4: Circular dichroism spectra of BLG mutants in the far- (left) and near-UV (right) in 10 mM sodium phosphate, pH 6.7 (A and B) and 10 mM glycine-HCl, pH 2.0 (C and D).

FIGURE 5: WT-BLG titration with retinol (A) and PA (B). Ri: [ligand] / [protein] ratio.
Circles: experimental data; line: non-linear fit. As BLG fluorescence decreases upon titration
with retinol, experimental data obtained with this ligand were expressed as an increase of fluorescence before mathematical analysis (calculated fluorescence intensity = 1-experimental fluorescence intensity).

FIGURE 6: Mass spectra of BLG - ligand complexes. Graph abscises are expressed as m/z ratio; numbers indicated on the graphs correspond to absolute masses (in Da). A: BLG + retinol. B: enlargement of the 9+ peak from A. The peak corresponding to a mass of 18,362 Da is that of BLG monomer and the one at 18,648 Da corresponds to BLG + retinol (286 Da). No signal for BLG dimer complexed with retinol could be detected because of too low intensity. C: BLG + PA. the peak annotated "+ 255 Da" corresponds to the BLG - PA complex. D: enlargement of the 13+ and 14+ peaks from C. The peaks corresponding to masses of 36,979 and 37,235 Da are those of BLG dimer complexed with 1 and 2 molecules of PA, respectively.

FIGURE 7: Interaction energy calculated from molecular modeling as a function of Log(K<sub>d</sub>) for native and all mutant BLG. A: retinol; B: PA.

FIGURE 8: Superimposition of the modeled structures for native and all BLG mutants complexed with retinol. The atoms are colored as in Figure 1.

FIGURE 9: Superimposition of the modeled structures of native and all BLG mutants complexed with PA (A). B to G: structure of PA complexed with K69R, K69N, A86Q, K69R-A86Q, K69N-A86Q and K69Y-A86H mutants, respectively. The mutated residues are indicated on each figure. The atoms are colored as in Figure 1. White sticks represent the H-bonds.
<figure><img src="image_1.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 8</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 9</figcaption></figure>
Design of β-Lactoglobulins with Increased Affinities for Retinol and Palmitic Acid

Damien Jayat, Jean-Charles Gaudin, Michèle Dalgalarrondo, Yvan Choiset, Jean-Marc Chobert, Vinh Tran, Carl Holt, Lindsay Sawyer, and Thomas Haertlé

<figure><img src="image_10.png" /></figure>