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The effect of conserved residue charge reversal on the folding of recombinant non-phosphorylated human β-casein
ABB Archives of Biochemistry and Biophysics 419 (2003) 244–250 www.elsevier.com/locate/yabbi
The effect of conserved residue charge reversal on the folding of recombinant non-phosphorylated human b-casein Hongyin Bu,1 Satish M. Sood, and Charles W. Slattery* Department of Biochemistry and Microbiology, Biochemistry Division, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA Received 25 June 2003, and in revised form 29 August 2003
Abstract A short stretch of 13 amino acids in the central portion of human b-casein contains four positively charged conserved residues, three Lys and one Arg. We changed these individually to Glu, reversing their charge, and compared the resulting recombinant proteins to the wild-type recombinant, monitoring thermal aggregation with turbidity as well as using the fluorescence of the intrinsic Trp, of hydrophobically bound ANS and fluorescence resonance energy transfer from Trp to ANS to detect differences in structure. The results demonstrate the need to maintain the actual or functional identity of these conserved charged amino acid residues in order to attain the protein folding and functional properties of the wild-type human b-casein molecule. They emphasize the probability that native human b-casein has a unique folding pattern that is important for its function of suspending minerals and delivering the protein and minerals to the neonate in a readily ingestible form. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Human b-casein; Conserved residues; Protein folding; Intrinsic and extrinsic fluorescence intensity; Fluorescence resonance energy transfer
The major casein (CN) protein in human milk is bCN, which exists at various levels of phosphorylation from zero to five [1]. These molecules associate and bind minerals, particularly Caþ2 and phosphate, and would precipitate were it not for the stabilizing ability of glycosylated j-CN in the system to suspend the casein– mineral complexes as colloidal milk micelles and thus deliver them to the neonate in a readily ingestible form. Once ingested, digestive enzymes must then be easily able to attack the micelles. It has long been debated as to whether a particular structure for b-CN is responsible for these different functions. Early physical measurements indicated very little secondary structure [2] and it and the other casein molecules are often considered to be random coil or unfolded proteins [3,4]. It is now thought that even though small portions of the chain may exhibit some thermostable secondary structure [5], * Corresponding author. Fax: 1-909-558-4887. E-mail address: [email protected] (C.W. Slattery). 1 Present address: Department of Pathology, School of Medicine, Washington University, St. Louis, MO 63110, USA.
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.08.032
different portions of the chains are held together in a noncooperative fashion to form a tertiary structure that is essentially random coil or rheomorphic and easily changed by interactions with other proteins or even ions in solution [6]. However, we have isolated the six phosphorylated forms of human b-CN from motherÕs milk and studied their association properties as the temperature increased from 4 to 37 °C [7–12]. We found that in the absence of Caþ2 ions, each of these, including the non-phosphorylated form (b-CN-0P), was monomeric at 4 °C and aggregated as the temperature increased, as measured by the turbidity of the solution. Furthermore, the aggregation was completely reversible upon cooling. Similar results were seen with bovine bCN [13,14]. This reversibility, along with other data on possible structure [3,15–19], strongly suggests that the molecule has a folding pattern that is relatively stable. Recently, we successfully expressed human b-CN-0P in Escherichia coli and found that either with four extra N-terminal amino acids [20] or with the exact native sequence (wild-type or WT) [21], the thermal aggregation pattern was no longer reversible. We see this as
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evidence for a unique folding pattern in the native protein that is altered by the harsh conditions of formation in and harvesting from inclusion bodies in E. coli. At protein concentrations where b-CN-0P did not aggregate, intrinsic fluorescence intensity of the single tryptophan residue, extrinsic fluorescence intensity of hydrophobically bound 8-anilino-1-naphthalene sulfonic acid (ANS),2 and fluorescence resonance energy transfer (FRET) from the Trp to the ANS were determined at different temperatures to document that there were significant differences in structure between the native and recombinant protein [21]. In this paper, we have begun to probe possible reasons for these differences by selecting certain charged amino acids that are conserved among most mammalian species [22,17] and reversing the charge by site-directed mutagenesis.
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by ion-exchange chromatography on Mono Q (Pharmacia) using a sodium chloride gradient [21]. The purified recombinant WT b-CN-0P and the mutants m1, m2, m3, and m4 were dialyzed against low salt buffer (0.02 M NaCl, 0.01 M imidizole, pH 7.0) for turbidity or Hepes (4-(2-hydroxyethyl) piperazine-1ethanesulfonic acid) buffer (10 mM Hepes, 50 mM NaCl, pH 7.4) for fluorescence studies and concentrated using the Slide-A-Lyzer (Pierce Biotechnology, Rockford, IL) concentrating system to above 3 mg/ml. They were then subjected to various physical measurements as the temperature was increased from about 4 to 37 °C. These included turbidity, intrinsic fluorescence intensity of the single Trp residue at position 154, external fluorescence intensity of bound ANS, and FRET between the Trp and ANS as described earlier [21]. Turbidity studies
Materials and methods The reagents used in these studies, unless otherwise noted, were of the highest grade obtainable from either Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). The cDNA for human b-CN encodes a precursor human b-CN of 226 amino acid residues (GenBank Accession No.: NP001882) [23–26]. The leader sequence contains 15 amino acids, which are removed during the maturation process. The WT recombinant human b-CN-0P corresponding to the remaining 211 residues was prepared as described previously [20,21]. This human b-CN has 45–62% homology with bovine, ovine, rat, and mouse b-CN [24]. As noted by Javor et al. [17], there are a number of positively charged Lys residues and an Arg residue that are conserved among six mammalian species for which the sequences are known [22], suggesting that they may play some part in the structure and function of the molecule. Those that we chose to change to Glu residues are all in the same region. The oligonucleotide primers, needed to obtain these mutations with the QuikChange Site-directed Mutagenesis Kit from Stratagene Inc. (La Jolla, CA), were synthesized in the Loma Linda University Core Facility in the Center for Molecular Biology and Gene Therapy. Sequencing of the gene, also in the Core Facility, showed that the correct mutations would be obtained. We thus produced four single amino acid mutants, designated m1 (K88E), m2 (K90E), m3 (K96E), and m4 (R98E). These proteins were each prepared like the WT recombinant in that they contained a traditional 6 His-tag to bind to Ni– NTA resin (Qiagen, Valencia, CA) for purification, a Factor Xa cleavage site at the N-terminus of the mature b-CN-0P, and after cleavage each was purified further 2 Abbreviations used: ANS, 8-anilino-1-naphthalene sulfonic acid; FRET, fluorescence resonance energy transfer.
Three mg/ml of b-CN has been successfully used in our laboratory to monitor the self-association by turbidity changes. All the recombinant protein samples were prepared at 3 mg/ml. A Perkin Elmer Lambda 2 UV/VIS spectrophotometer attached to a temperature controller was used for the measurements. Absorbances at 400 nm were determined at different temperature points from 4 to 37 °C with 3 °C increments. When the temperature reached the desired point, the absorbance was read after the samples were equilibrated for 30 min at the given temperature. After the reading at the maximum temperature, the sample was removed from the spectrophotometer and kept on ice until it became transparent. Then, it was removed from the cuvette and stored at 4 °C until the next experiment. A second temperature cycle was then performed. Steady state fluorescence spectroscopy The purpose of these studies was to investigate the changes in the human b-CN structure caused by temperature that are the major factors involved in the protein association. Intrinsic fluorescence is generated from Trp residues upon light excitation and any change in environment, perhaps from protein conformational change, affects the fluorescence. An excitation wavelength of 295 nm is often used to avoid excitation of Tyr residues. The mutant samples were diluted to 50 lg/ml (approximately 2 lM) in 10 mM Hepes buffer at pH 7.4 and spectra were measured in a cell of 1 cm path length as described previously for native and recombinant wildtype non-phosphorylated human b-CN [21]. The binding of a fluorescent dye, 1-anilino-naphthalene-8-sulfonate (ANS), as well as fluorescence resonance energy transfer (FRET) from the Trp to the ANS in the b-CN mutants were also monitored spectrophotometrically as a function of temperature as previously
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described [21]. The ANS concentration was 80 lM in each case for a dye:protein ratio of 40.
Results Turbidity measurements As was shown earlier, the WT recombinant human bCN-0P has a temperature-dependent pattern of turbidity increase that is similar to the native protein during the first thermal cycle but which is irreproducible upon subsequent thermal cycling [21]. The turbidity patterns for the first and second thermal cycles for each chargereversed mutant are shown in Fig. 1. As with the WT recombinant protein [21], the first-cycle patterns of protein aggregation for the mutants were not reproducible after cooling. This may be due to the proteins still being partially aggregated, even after prolonged exposure to 4 °C, resulting in an altered pattern of further aggregation. Alternatively, the aggregation in the first thermal cycle could change the protein folding and result in a different aggregation pattern for subsequent cycles. The first-cycle pattern was reproduced by treatment with a high concentration of urea followed by dialysis against buffer [21]. The degree of deviation from the first-cycle was least for m4 and greatest for m2 while the m1 mutant appeared to be most like the WT protein in this respect. None of these mutants actually repaired the irreversibility problem, probably the result of misfolding, seen with WT. It is, however, interesting that these charged residues, predicted to be exposed to the molecular surface to function only in solubilizing the
molecule in an aqueous solution, nevertheless have an effect on how the molecules aggregate through hydrophobic interactions as the temperature is increased. To better compare the mutants and the WT protein under relatively standard conditions, we chose to look at their properties only during a first thermal cycle. During the first temperature cycle, the mutants all show an aggregation pattern that is different from the WT protein (Fig. 2). The K-E mutants, m1, m2, and m3, aggregate earlier than WT and reach a higher level of aggregation at 37 °C. The m1 and m3 mutants may be slightly aggregated even at 4 °C while the m4 R-E mutant aggregates less readily as the temperature is increased, but achieves near equivalence with WT at 37 °C. Possible reasons for these differences were probed with fluorescence measurements. Intrinsic tryptophan fluorescence Tryptophan fluorescence spectra for the charge-reversed single amino acid mutants of human b-CN-0P are compared in Fig. 3 at 4, 22, and 37 °C. The spectra are different from those of the WT or native b-CN-0P. The fluorescence emission of m1 and m3 showed decreases in intensity with temperature but no wavelength shift to a shorter wavelength. The fluorescence emission maxima at 4, 22, and 37 °C were 360, 362, and 361 nm for m1 and 356, 356, and 358 nm for m3. The fluorescence emission spectra of the m2 and m4 mutants are qualitatively similar to the spectrum of the WT recombinant human b-CN-0P in that their fluorescence intensity decreases as the temperature changes from 4 to 37 °C and there are slight wavelength shifts at 37 °C.
Fig. 1. The turbidity (OD400 ) as a function of temperature on the first thermal cycle (r) and the second thermal cycle (j) for m1 (K88E), m2 (K90E), m3 (K96E), and m4 (R98E) recombinant human b-casein molecules. The protein concentrations were at 3 mg/ml.
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Fig. 2. A comparison of the turbidity (OD400 ) as a function of temperature during the first thermal cycle for wild-type (WT), and the four chargereversed human b-casein mutants. The protein concentrations were at 3 mg/ml.
Fig. 3. The fluorescence intensity spectra of the intrinsic tryptophan for the four charge-reversed human b-casein mutants at 4, 22, and 37 °C. The protein concentrations were at 50 lg/ml and excitation was at 295 nm.
The emission maxima at 4, 22, and 37 °C are 356, 357, and 352 nm for m2 and 355, 352, and 351 nm for m4. ANS fluorescence ANS fluorescence emission spectra of the single amino acid mutants of human b-CN are shown in Fig. 4. The intensity of ANS fluorescence emission for m1 increased as the temperature increased from 4 to 37 °C. The emission maximum showed no blue shift when the temperature increased to 22 and 37 °C. The fluorescence
emission maxima of m1 at 4, 22, and 37 °C were 519, 519, and 518 nm, respectively. The intensity of ANS fluorescence emission for m2 was decreased when temperature increased from 4 to 22 °C, and then increased when the temperature reached 37 °C. The fluorescence emission maxima of m2 at 4, 22, and 37 °C were 505, 505, and 502, respectively. The ANS fluorescence emission of m3 showed decreased intensity when temperature increased from 4 to 37 °C. The emission maxima were 513, 511, and 509 nm at 4, 22, and 37 °C, respectively. The ANS fluorescence
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Fig. 4. The fluorescence intensity spectra of externally bound ANS for the four charge-reversed human b-casein mutants at 4, 22, and 37 °C. The protein concentrations were at 50 lg/ml and the ANS concentration was 80 lM. Excitation to measure ANS was at 365 nm.
Fig. 5. Fluorescence resonance energy transfer (FRET) from tryptophan to ANS for the four charge-reversed human b-casein mutants at 4, 22, and 37 °C. The protein concentrations were at 50 lg/ml and the ANS concentration was 80 lM. Excitation for tryptophan was at 295 nm and its fluorescence emission around 350 nm was the excitation wavelength for ANS emission.
of m4 showed no significant change in intensity as the temperature increased from 4 to 37 °C. The fluorescence emission maxima at 4, 22, and 37 °C were 505, 505, and 494 nm, respectively.
had significant Trp to ANS transfer at 37 °C (Fig. 5). This indicates that m2 had a significant structural change when the temperature increased from 22 to 37 °C.
Fluorescence resonance energy transfer
Discussion
None of the four single amino acid mutants of b-CN0P showed significant FRET at 4 or 22 °C and only m2
An examination of the amino acid sequence of human b-CN shows why little secondary structure is seen.
H. Bu et al. / Archives of Biochemistry and Biophysics 419 (2003) 244–250
Except for a few notable regions, there is a rather uniform distribution of Pro residues, 9 of 37 of which are conserved [17], that tend to disrupt a-helix and b-strand formation. The 40 N-terminal residues until the first Pro is encountered comprise an ‘‘acidic peptide’’ containing 12–19 negative charges, depending upon the level of phosphorylation, and only about 6 positive charges. The phosphorylated Ser or Thr residues, phosphorylated in order at positions 10 or 9, 9 or 10, 8, 6, and 3 [27], are the major Caþ2 binding sites. There is an 18-residue region between Pro residues near the C-terminus, from residues 178–195, that is mostly hydrophobic. There is a 13-residue region from residues 88–100 and a 10-residue region from residues 117–126, each of which begins with a conserved Lys [17]. Aside from these, the gaps between Pro residues vary only from zero to five. The 13-residue region contains seven residues that are actually or functionally identical among species and the following Pro residue is also conserved [17], suggesting that this region is important for protein structure and function. Four of the seven conserved residues are positively charged. It is possible that these residues could be involved in the formation of the structure directing the self-association of human b-CN molecules. The charged residues are close to one another and would be expected to be in a solvent-accessible turn. Such a turn was postulated by Kumosinski et al. [28] in the homologous region of bovine b-CN for their three-dimensional molecular model. The proximity of opposite or like charges could impose constraints on this turn and affect the chain folding. With this in mind, we individually reversed the charge of these residues by site-directed mutagenesis, expressed and purified the proteins from E. coli, and subjected them to various physical measurements for comparison with each other and the WT recombinant protein. Mutant m1 (K88E) The Lys residue at position 88 is close to another at residue 90 and also near negative charges at residues 82 and 85. Changing it to a negatively charged Glu thus has the potential to disrupt peptide structure in this region and influence folding of the remainder of the chain and subsequent self-association. Alternatively, protein structure could remain the same but intermolecular charge–charge interactions could influence self-association. Turbidity measurements (Fig. 2) indeed show that this mutant begins self-association at a lower temperature than the WT or the other mutants and achieves the highest state of aggregation at 37 °C. Although this could be due to altered intermolecular charge-charge interactions, there is also a difference in protein structure. The fluorescence intensity of the intrinsic Trp 154 is about at the level of the native b-CN-0P and higher than the WT protein [21]. The decrease in intensity with
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temperature indicates that the Trp is in a polar environment. However, the blue shift with increased temperature for the maxima of the spectra, seen with the b-CN-0P and WT proteins [21], has been abolished. This indicates that with m1, there is little conformational change with temperature to change the polarity of the environment near Trp 154. The lowered ANS fluorescence (Fig. 4) and the lack of significant FRET (Fig. 5) also suggest that the K88E mutation tends to lock the molecule into a particular conformation with less hydrophobic surface and makes it somewhat inflexible as the temperature is changed. Mutant m2 (K90E) This mutation places two negative charges at positions 90 and 91 adjacent to each other. The molecule appears to fold into a conformation with properties somewhat intermediate between WT and native b-CN0P, although it still shows the irreversibility of thermal aggregation (Fig. 1). The first-cycle aggregation pattern (Fig. 2) suggests that it may have more interactive surface hydrophobic groups than the WT protein. However, because of the higher fluorescence intensity for m2 (Fig. 4) than for WT [21], it seems likely that Trp154 is more exposed to the solvent. Furthermore, the blue shift for the maxima indicates more exposure to the solvent as the temperature is increased. It is interesting that while there is a difference in the temperature-dependence of ANS fluorescence (Fig. 4), suggesting a more fixed structure at lower temperatures for m2 than WT with a major change only above 22 °C for m2, the ANS fluorescence intensity at 37 °C is only slightly higher. This is probably due to there being only a fixed number of ANS binding sites in each that limit the fluorescence. The results of FRET (Fig. 5) correspond with this, since the higher intensities of Trp fluorescence for m2 only cause levels of energy transfer and secondary ANS fluorescence slightly above those for the WT protein [21]. Mutants m3 (K96E) and m4 (R98E) Since these two positive charges are close together and separated only by a Gly residue, it might be expected that changing either one to a negative charge would have a similar effect, and such is the case. An electrostatic attraction in this region could cause an entirely different peptide conformation than electrostatic repulsion of the like charges. In any case, the irreversibility of thermal aggregation of both is diminished over WT, m1 or m2 (Fig. 1) and, although the first-cycle aggregation patterns are a bit different, they approach that of the WT protein (Fig. 2). The Trp154 fluorescence intensity spectra are very similar in magnitude and in temperature-dependence with the blue shift being abolished (Fig. 3), indicating that the Trp154 must be in
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about the same relatively polar environment in each of these and also in m1 and they are all somewhat inflexible. The ANS fluorescence spectra of m3 show more temperature-dependence than for m4 but both are higher in magnitude than m1 and slightly lower than m2 (Fig. 4). The FRET results (Fig. 5) also show a bit more flexibility for m3 than for either m4 or m1. There is evidence to suggest that this cluster of positively charged residues should be placed not only on the surface of the individual b-CN molecules but also at the surface of the micelles. It has been reported [29] that the enzyme plasmin in human milk will cleave the b-CN molecule at K96 and R98 in a similar fashion to such cleavage in bovine milk [30]. It was noted by Farrell et al. [31] that bovine j-CN also contains a cluster of positively charged residues that are susceptible to protease action in isolation but not as readily in the micelle. It was postulated [31] that the cluster in b-CN is more readily accessible in the micelle and thus competes to protect the j-CN from proteolysis, which would result in premature coagulation of the milk micelles. Thus, even if these conserved residues are not responsible for the unique folding of the b-CN molecule in a major way, they may have another important function and other residues may direct the folding of the molecule in order to maintain that function. Summary and conclusions The m1, m3, and m4 charge-reversed mutants of the four positively charged amino acid residues near the mid-region of human b-CN-0P significantly altered the turbidity, Trp intrinsic fluorescence, extrinsic fluorescence of added ANS, and Trp to ANS fluorescence resonance energy transfer relative to the recombinant WT protein. The reversal of a charge may alter the structure of this region because of different charge– charge interactions and subsequently affect the molecular folding of the entire molecule. On the other hand, the m2 mutation has properties that are much like those of the WT protein. This suggests that the structure and environment of Lys90 may already be such that the change in the charge does not significantly affect the structure at that point or the subsequent folding of the molecule. It is probably exposed to the solvent and could possibly be affected by substitution of a non-polar amino acid. The results of this study demonstrate the need to maintain the actual or functional identity of these conserved charged amino acid residues in order to attain the protein folding and/or functional properties of the wildtype human b-CN-0P molecule. They emphasize the probability that b-CN has a unique folding pattern that is important for its function of suspending minerals and delivering the protein and minerals to the neonate in a readily ingestible form.
Acknowledgments The authors wish to thank Dr. Aladar Szalay for providing access to the fluorescence spectrophotometer in his laboratory for this study.
References [1] R. Greenberg, M.L. Groves, J. Dairy Res. 46 (1979) 235–239. [2] T.T. Herskovits, Biochemistry 5 (1966) 1018–1026. [3] C. Holt, L. Sawyer, J. Chem. Soc., Faraday Trans. 199 (1993) 2683–2692. [4] J. Bhattacharyya, K.P. Das, J. Biol. Chem. 274 (1999) 15505– 15509. [5] H.M. Farrell Jr., P.X. Qi, E.D. Wickham, J.J. Unruh, J. Protein Chem. 21 (2002) 307–321. [6] C.D. Syme, E.W. Blanch, C. Holt, R. Jakes, M. Goedert, L. Hecht, L.D. Barron, Eur. J. Biochem. 269 (2002) 148–156. [7] S.M. Sood, P. Chang, C.W. Slattery, Arch. Biochem. Biophys. 242 (1985) 355–364. [8] S.M. Sood, P. Chang, C.W. Slattery, Arch. Biochem. Biophys. 264 (1988) 574–583. [9] S.M. Sood, P. Chang, C.W. Slattery, Arch. Biochem. Biophys. 277 (1990) 415–421. [10] S.M. Sood, P. Chang, C.W. Slattery, J. Dairy Sci. 75 (1992) 2937– 2945. [11] S.M. Sood, C.W. Slattery, J. Dairy Sci. 77 (1994) 405–412. [12] S.M. Sood, C.W. Slattery, J. Dairy Sci. 80 (1997) 1554–1560. [13] D.F. Waugh, L.K. Creamer, C.W. Slattery, G.W. Dresdner, Biochemistry 9 (1970) 786–795. [14] S. Arima, R. Niki, K. Takase, J. Dairy Res. 46 (1979) 281–282. [15] J. Garnier, J. Mol. Biol. 19 (1966) 586–590. [16] L.C. Chaplin, D.C. Clark, L.J. Smith, Biochim. Biophys. Acta 956 (1988) 162–172. [17] G.T. Javor, S.M. Sood, P. Chang, C.W. Slattery, Arch. Biochem. Biophys. 289 (1991) 39–46. [18] P.W. Caessens, H.H. DeJongh, W. Norde, H. Gruppen, Biochim. Biophys. Acta 1430 (1999) 73–83. [19] H.M. Farrell Jr., E.D. Wickham, J.J. Unruh, P.X. Qi, P.D. Hoagland, Food Hydrocolloids 15 (2001) 341–354. [20] Y. Hu, S.M. Sood, C.W. Slattery, Arch. Biochem. Biophys. 383 (2000) 215–224. [21] H. Bu, Y. Hu, S.M. Sood, C.W. Slattery, Arch. Biochem. Biophys. 415 (2003) 213–220. [22] C. Holt, L. Sawyer, Protein Eng. 2 (1988) 251–259. [23] R.S. Menon, R.G. Ham, Nucleic Acids Res. 17 (1989) 2869. [24] B. Lonnerdal, S. Bergstrom, Y. Andersson, K. Hjalmarsson, A. Sundqvist, O. Hernell, FEBS Lett. 269 (1990) 153–156. [25] L. Hansson, A. Edlund, T. Johansson, O. Hernell, M. Stromqvist, S. Lindquist, B. Lonnerdal, S. Bergstrom, Gene 139 (1994) 193– 199. [26] R. Greenberg, M.L. Groves, H.J. Dower, J. Biol. Chem. 259 (1984) 5132–5138. [27] E.W. Bingham, H.M. Farrell Jr., J. Dairy Sci. 60 (1977) 1199– 1207. [28] T.F. Kumosinski, E.M. Brown, H.M. Farrell Jr., J. Dairy Sci. 76 (1993) 931–945. [29] R. Greenberg, M.L. Groves, Biochem. Biophys. Res. Commun. 125 (1984) 463–468. [30] W.N. Eigel, Int. J. Biochem. 8 (1977) 187–192. [31] H.M. Farrell Jr., E.D. Wickham, H.J. Dower, E.G. Piotrowski, P.D. Hoagland, P.H. Cooke, M.L. Groves, J. Protein Chem. 18 (1999) 637–652.
The effect of conserved residue charge reversal on the folding of recombinant non-phosphorylated human b-casein Hongyin Bu,1 Satish M. Sood, and Charles W. Slattery* Department of Biochemistry and Microbiology, Biochemistry Division, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA Received 25 June 2003, and in revised form 29 August 2003
Abstract A short stretch of 13 amino acids in the central portion of human b-casein contains four positively charged conserved residues, three Lys and one Arg. We changed these individually to Glu, reversing their charge, and compared the resulting recombinant proteins to the wild-type recombinant, monitoring thermal aggregation with turbidity as well as using the fluorescence of the intrinsic Trp, of hydrophobically bound ANS and fluorescence resonance energy transfer from Trp to ANS to detect differences in structure. The results demonstrate the need to maintain the actual or functional identity of these conserved charged amino acid residues in order to attain the protein folding and functional properties of the wild-type human b-casein molecule. They emphasize the probability that native human b-casein has a unique folding pattern that is important for its function of suspending minerals and delivering the protein and minerals to the neonate in a readily ingestible form. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Human b-casein; Conserved residues; Protein folding; Intrinsic and extrinsic fluorescence intensity; Fluorescence resonance energy transfer
The major casein (CN) protein in human milk is bCN, which exists at various levels of phosphorylation from zero to five [1]. These molecules associate and bind minerals, particularly Caþ2 and phosphate, and would precipitate were it not for the stabilizing ability of glycosylated j-CN in the system to suspend the casein– mineral complexes as colloidal milk micelles and thus deliver them to the neonate in a readily ingestible form. Once ingested, digestive enzymes must then be easily able to attack the micelles. It has long been debated as to whether a particular structure for b-CN is responsible for these different functions. Early physical measurements indicated very little secondary structure [2] and it and the other casein molecules are often considered to be random coil or unfolded proteins [3,4]. It is now thought that even though small portions of the chain may exhibit some thermostable secondary structure [5], * Corresponding author. Fax: 1-909-558-4887. E-mail address: [email protected] (C.W. Slattery). 1 Present address: Department of Pathology, School of Medicine, Washington University, St. Louis, MO 63110, USA.
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.08.032
different portions of the chains are held together in a noncooperative fashion to form a tertiary structure that is essentially random coil or rheomorphic and easily changed by interactions with other proteins or even ions in solution [6]. However, we have isolated the six phosphorylated forms of human b-CN from motherÕs milk and studied their association properties as the temperature increased from 4 to 37 °C [7–12]. We found that in the absence of Caþ2 ions, each of these, including the non-phosphorylated form (b-CN-0P), was monomeric at 4 °C and aggregated as the temperature increased, as measured by the turbidity of the solution. Furthermore, the aggregation was completely reversible upon cooling. Similar results were seen with bovine bCN [13,14]. This reversibility, along with other data on possible structure [3,15–19], strongly suggests that the molecule has a folding pattern that is relatively stable. Recently, we successfully expressed human b-CN-0P in Escherichia coli and found that either with four extra N-terminal amino acids [20] or with the exact native sequence (wild-type or WT) [21], the thermal aggregation pattern was no longer reversible. We see this as
H. Bu et al. / Archives of Biochemistry and Biophysics 419 (2003) 244–250
evidence for a unique folding pattern in the native protein that is altered by the harsh conditions of formation in and harvesting from inclusion bodies in E. coli. At protein concentrations where b-CN-0P did not aggregate, intrinsic fluorescence intensity of the single tryptophan residue, extrinsic fluorescence intensity of hydrophobically bound 8-anilino-1-naphthalene sulfonic acid (ANS),2 and fluorescence resonance energy transfer (FRET) from the Trp to the ANS were determined at different temperatures to document that there were significant differences in structure between the native and recombinant protein [21]. In this paper, we have begun to probe possible reasons for these differences by selecting certain charged amino acids that are conserved among most mammalian species [22,17] and reversing the charge by site-directed mutagenesis.
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by ion-exchange chromatography on Mono Q (Pharmacia) using a sodium chloride gradient [21]. The purified recombinant WT b-CN-0P and the mutants m1, m2, m3, and m4 were dialyzed against low salt buffer (0.02 M NaCl, 0.01 M imidizole, pH 7.0) for turbidity or Hepes (4-(2-hydroxyethyl) piperazine-1ethanesulfonic acid) buffer (10 mM Hepes, 50 mM NaCl, pH 7.4) for fluorescence studies and concentrated using the Slide-A-Lyzer (Pierce Biotechnology, Rockford, IL) concentrating system to above 3 mg/ml. They were then subjected to various physical measurements as the temperature was increased from about 4 to 37 °C. These included turbidity, intrinsic fluorescence intensity of the single Trp residue at position 154, external fluorescence intensity of bound ANS, and FRET between the Trp and ANS as described earlier [21]. Turbidity studies
Materials and methods The reagents used in these studies, unless otherwise noted, were of the highest grade obtainable from either Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). The cDNA for human b-CN encodes a precursor human b-CN of 226 amino acid residues (GenBank Accession No.: NP001882) [23–26]. The leader sequence contains 15 amino acids, which are removed during the maturation process. The WT recombinant human b-CN-0P corresponding to the remaining 211 residues was prepared as described previously [20,21]. This human b-CN has 45–62% homology with bovine, ovine, rat, and mouse b-CN [24]. As noted by Javor et al. [17], there are a number of positively charged Lys residues and an Arg residue that are conserved among six mammalian species for which the sequences are known [22], suggesting that they may play some part in the structure and function of the molecule. Those that we chose to change to Glu residues are all in the same region. The oligonucleotide primers, needed to obtain these mutations with the QuikChange Site-directed Mutagenesis Kit from Stratagene Inc. (La Jolla, CA), were synthesized in the Loma Linda University Core Facility in the Center for Molecular Biology and Gene Therapy. Sequencing of the gene, also in the Core Facility, showed that the correct mutations would be obtained. We thus produced four single amino acid mutants, designated m1 (K88E), m2 (K90E), m3 (K96E), and m4 (R98E). These proteins were each prepared like the WT recombinant in that they contained a traditional 6 His-tag to bind to Ni– NTA resin (Qiagen, Valencia, CA) for purification, a Factor Xa cleavage site at the N-terminus of the mature b-CN-0P, and after cleavage each was purified further 2 Abbreviations used: ANS, 8-anilino-1-naphthalene sulfonic acid; FRET, fluorescence resonance energy transfer.
Three mg/ml of b-CN has been successfully used in our laboratory to monitor the self-association by turbidity changes. All the recombinant protein samples were prepared at 3 mg/ml. A Perkin Elmer Lambda 2 UV/VIS spectrophotometer attached to a temperature controller was used for the measurements. Absorbances at 400 nm were determined at different temperature points from 4 to 37 °C with 3 °C increments. When the temperature reached the desired point, the absorbance was read after the samples were equilibrated for 30 min at the given temperature. After the reading at the maximum temperature, the sample was removed from the spectrophotometer and kept on ice until it became transparent. Then, it was removed from the cuvette and stored at 4 °C until the next experiment. A second temperature cycle was then performed. Steady state fluorescence spectroscopy The purpose of these studies was to investigate the changes in the human b-CN structure caused by temperature that are the major factors involved in the protein association. Intrinsic fluorescence is generated from Trp residues upon light excitation and any change in environment, perhaps from protein conformational change, affects the fluorescence. An excitation wavelength of 295 nm is often used to avoid excitation of Tyr residues. The mutant samples were diluted to 50 lg/ml (approximately 2 lM) in 10 mM Hepes buffer at pH 7.4 and spectra were measured in a cell of 1 cm path length as described previously for native and recombinant wildtype non-phosphorylated human b-CN [21]. The binding of a fluorescent dye, 1-anilino-naphthalene-8-sulfonate (ANS), as well as fluorescence resonance energy transfer (FRET) from the Trp to the ANS in the b-CN mutants were also monitored spectrophotometrically as a function of temperature as previously
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described [21]. The ANS concentration was 80 lM in each case for a dye:protein ratio of 40.
Results Turbidity measurements As was shown earlier, the WT recombinant human bCN-0P has a temperature-dependent pattern of turbidity increase that is similar to the native protein during the first thermal cycle but which is irreproducible upon subsequent thermal cycling [21]. The turbidity patterns for the first and second thermal cycles for each chargereversed mutant are shown in Fig. 1. As with the WT recombinant protein [21], the first-cycle patterns of protein aggregation for the mutants were not reproducible after cooling. This may be due to the proteins still being partially aggregated, even after prolonged exposure to 4 °C, resulting in an altered pattern of further aggregation. Alternatively, the aggregation in the first thermal cycle could change the protein folding and result in a different aggregation pattern for subsequent cycles. The first-cycle pattern was reproduced by treatment with a high concentration of urea followed by dialysis against buffer [21]. The degree of deviation from the first-cycle was least for m4 and greatest for m2 while the m1 mutant appeared to be most like the WT protein in this respect. None of these mutants actually repaired the irreversibility problem, probably the result of misfolding, seen with WT. It is, however, interesting that these charged residues, predicted to be exposed to the molecular surface to function only in solubilizing the
molecule in an aqueous solution, nevertheless have an effect on how the molecules aggregate through hydrophobic interactions as the temperature is increased. To better compare the mutants and the WT protein under relatively standard conditions, we chose to look at their properties only during a first thermal cycle. During the first temperature cycle, the mutants all show an aggregation pattern that is different from the WT protein (Fig. 2). The K-E mutants, m1, m2, and m3, aggregate earlier than WT and reach a higher level of aggregation at 37 °C. The m1 and m3 mutants may be slightly aggregated even at 4 °C while the m4 R-E mutant aggregates less readily as the temperature is increased, but achieves near equivalence with WT at 37 °C. Possible reasons for these differences were probed with fluorescence measurements. Intrinsic tryptophan fluorescence Tryptophan fluorescence spectra for the charge-reversed single amino acid mutants of human b-CN-0P are compared in Fig. 3 at 4, 22, and 37 °C. The spectra are different from those of the WT or native b-CN-0P. The fluorescence emission of m1 and m3 showed decreases in intensity with temperature but no wavelength shift to a shorter wavelength. The fluorescence emission maxima at 4, 22, and 37 °C were 360, 362, and 361 nm for m1 and 356, 356, and 358 nm for m3. The fluorescence emission spectra of the m2 and m4 mutants are qualitatively similar to the spectrum of the WT recombinant human b-CN-0P in that their fluorescence intensity decreases as the temperature changes from 4 to 37 °C and there are slight wavelength shifts at 37 °C.
Fig. 1. The turbidity (OD400 ) as a function of temperature on the first thermal cycle (r) and the second thermal cycle (j) for m1 (K88E), m2 (K90E), m3 (K96E), and m4 (R98E) recombinant human b-casein molecules. The protein concentrations were at 3 mg/ml.
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Fig. 2. A comparison of the turbidity (OD400 ) as a function of temperature during the first thermal cycle for wild-type (WT), and the four chargereversed human b-casein mutants. The protein concentrations were at 3 mg/ml.
Fig. 3. The fluorescence intensity spectra of the intrinsic tryptophan for the four charge-reversed human b-casein mutants at 4, 22, and 37 °C. The protein concentrations were at 50 lg/ml and excitation was at 295 nm.
The emission maxima at 4, 22, and 37 °C are 356, 357, and 352 nm for m2 and 355, 352, and 351 nm for m4. ANS fluorescence ANS fluorescence emission spectra of the single amino acid mutants of human b-CN are shown in Fig. 4. The intensity of ANS fluorescence emission for m1 increased as the temperature increased from 4 to 37 °C. The emission maximum showed no blue shift when the temperature increased to 22 and 37 °C. The fluorescence
emission maxima of m1 at 4, 22, and 37 °C were 519, 519, and 518 nm, respectively. The intensity of ANS fluorescence emission for m2 was decreased when temperature increased from 4 to 22 °C, and then increased when the temperature reached 37 °C. The fluorescence emission maxima of m2 at 4, 22, and 37 °C were 505, 505, and 502, respectively. The ANS fluorescence emission of m3 showed decreased intensity when temperature increased from 4 to 37 °C. The emission maxima were 513, 511, and 509 nm at 4, 22, and 37 °C, respectively. The ANS fluorescence
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Fig. 4. The fluorescence intensity spectra of externally bound ANS for the four charge-reversed human b-casein mutants at 4, 22, and 37 °C. The protein concentrations were at 50 lg/ml and the ANS concentration was 80 lM. Excitation to measure ANS was at 365 nm.
Fig. 5. Fluorescence resonance energy transfer (FRET) from tryptophan to ANS for the four charge-reversed human b-casein mutants at 4, 22, and 37 °C. The protein concentrations were at 50 lg/ml and the ANS concentration was 80 lM. Excitation for tryptophan was at 295 nm and its fluorescence emission around 350 nm was the excitation wavelength for ANS emission.
of m4 showed no significant change in intensity as the temperature increased from 4 to 37 °C. The fluorescence emission maxima at 4, 22, and 37 °C were 505, 505, and 494 nm, respectively.
had significant Trp to ANS transfer at 37 °C (Fig. 5). This indicates that m2 had a significant structural change when the temperature increased from 22 to 37 °C.
Fluorescence resonance energy transfer
Discussion
None of the four single amino acid mutants of b-CN0P showed significant FRET at 4 or 22 °C and only m2
An examination of the amino acid sequence of human b-CN shows why little secondary structure is seen.
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Except for a few notable regions, there is a rather uniform distribution of Pro residues, 9 of 37 of which are conserved [17], that tend to disrupt a-helix and b-strand formation. The 40 N-terminal residues until the first Pro is encountered comprise an ‘‘acidic peptide’’ containing 12–19 negative charges, depending upon the level of phosphorylation, and only about 6 positive charges. The phosphorylated Ser or Thr residues, phosphorylated in order at positions 10 or 9, 9 or 10, 8, 6, and 3 [27], are the major Caþ2 binding sites. There is an 18-residue region between Pro residues near the C-terminus, from residues 178–195, that is mostly hydrophobic. There is a 13-residue region from residues 88–100 and a 10-residue region from residues 117–126, each of which begins with a conserved Lys [17]. Aside from these, the gaps between Pro residues vary only from zero to five. The 13-residue region contains seven residues that are actually or functionally identical among species and the following Pro residue is also conserved [17], suggesting that this region is important for protein structure and function. Four of the seven conserved residues are positively charged. It is possible that these residues could be involved in the formation of the structure directing the self-association of human b-CN molecules. The charged residues are close to one another and would be expected to be in a solvent-accessible turn. Such a turn was postulated by Kumosinski et al. [28] in the homologous region of bovine b-CN for their three-dimensional molecular model. The proximity of opposite or like charges could impose constraints on this turn and affect the chain folding. With this in mind, we individually reversed the charge of these residues by site-directed mutagenesis, expressed and purified the proteins from E. coli, and subjected them to various physical measurements for comparison with each other and the WT recombinant protein. Mutant m1 (K88E) The Lys residue at position 88 is close to another at residue 90 and also near negative charges at residues 82 and 85. Changing it to a negatively charged Glu thus has the potential to disrupt peptide structure in this region and influence folding of the remainder of the chain and subsequent self-association. Alternatively, protein structure could remain the same but intermolecular charge–charge interactions could influence self-association. Turbidity measurements (Fig. 2) indeed show that this mutant begins self-association at a lower temperature than the WT or the other mutants and achieves the highest state of aggregation at 37 °C. Although this could be due to altered intermolecular charge-charge interactions, there is also a difference in protein structure. The fluorescence intensity of the intrinsic Trp 154 is about at the level of the native b-CN-0P and higher than the WT protein [21]. The decrease in intensity with
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temperature indicates that the Trp is in a polar environment. However, the blue shift with increased temperature for the maxima of the spectra, seen with the b-CN-0P and WT proteins [21], has been abolished. This indicates that with m1, there is little conformational change with temperature to change the polarity of the environment near Trp 154. The lowered ANS fluorescence (Fig. 4) and the lack of significant FRET (Fig. 5) also suggest that the K88E mutation tends to lock the molecule into a particular conformation with less hydrophobic surface and makes it somewhat inflexible as the temperature is changed. Mutant m2 (K90E) This mutation places two negative charges at positions 90 and 91 adjacent to each other. The molecule appears to fold into a conformation with properties somewhat intermediate between WT and native b-CN0P, although it still shows the irreversibility of thermal aggregation (Fig. 1). The first-cycle aggregation pattern (Fig. 2) suggests that it may have more interactive surface hydrophobic groups than the WT protein. However, because of the higher fluorescence intensity for m2 (Fig. 4) than for WT [21], it seems likely that Trp154 is more exposed to the solvent. Furthermore, the blue shift for the maxima indicates more exposure to the solvent as the temperature is increased. It is interesting that while there is a difference in the temperature-dependence of ANS fluorescence (Fig. 4), suggesting a more fixed structure at lower temperatures for m2 than WT with a major change only above 22 °C for m2, the ANS fluorescence intensity at 37 °C is only slightly higher. This is probably due to there being only a fixed number of ANS binding sites in each that limit the fluorescence. The results of FRET (Fig. 5) correspond with this, since the higher intensities of Trp fluorescence for m2 only cause levels of energy transfer and secondary ANS fluorescence slightly above those for the WT protein [21]. Mutants m3 (K96E) and m4 (R98E) Since these two positive charges are close together and separated only by a Gly residue, it might be expected that changing either one to a negative charge would have a similar effect, and such is the case. An electrostatic attraction in this region could cause an entirely different peptide conformation than electrostatic repulsion of the like charges. In any case, the irreversibility of thermal aggregation of both is diminished over WT, m1 or m2 (Fig. 1) and, although the first-cycle aggregation patterns are a bit different, they approach that of the WT protein (Fig. 2). The Trp154 fluorescence intensity spectra are very similar in magnitude and in temperature-dependence with the blue shift being abolished (Fig. 3), indicating that the Trp154 must be in
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about the same relatively polar environment in each of these and also in m1 and they are all somewhat inflexible. The ANS fluorescence spectra of m3 show more temperature-dependence than for m4 but both are higher in magnitude than m1 and slightly lower than m2 (Fig. 4). The FRET results (Fig. 5) also show a bit more flexibility for m3 than for either m4 or m1. There is evidence to suggest that this cluster of positively charged residues should be placed not only on the surface of the individual b-CN molecules but also at the surface of the micelles. It has been reported [29] that the enzyme plasmin in human milk will cleave the b-CN molecule at K96 and R98 in a similar fashion to such cleavage in bovine milk [30]. It was noted by Farrell et al. [31] that bovine j-CN also contains a cluster of positively charged residues that are susceptible to protease action in isolation but not as readily in the micelle. It was postulated [31] that the cluster in b-CN is more readily accessible in the micelle and thus competes to protect the j-CN from proteolysis, which would result in premature coagulation of the milk micelles. Thus, even if these conserved residues are not responsible for the unique folding of the b-CN molecule in a major way, they may have another important function and other residues may direct the folding of the molecule in order to maintain that function. Summary and conclusions The m1, m3, and m4 charge-reversed mutants of the four positively charged amino acid residues near the mid-region of human b-CN-0P significantly altered the turbidity, Trp intrinsic fluorescence, extrinsic fluorescence of added ANS, and Trp to ANS fluorescence resonance energy transfer relative to the recombinant WT protein. The reversal of a charge may alter the structure of this region because of different charge– charge interactions and subsequently affect the molecular folding of the entire molecule. On the other hand, the m2 mutation has properties that are much like those of the WT protein. This suggests that the structure and environment of Lys90 may already be such that the change in the charge does not significantly affect the structure at that point or the subsequent folding of the molecule. It is probably exposed to the solvent and could possibly be affected by substitution of a non-polar amino acid. The results of this study demonstrate the need to maintain the actual or functional identity of these conserved charged amino acid residues in order to attain the protein folding and/or functional properties of the wildtype human b-CN-0P molecule. They emphasize the probability that b-CN has a unique folding pattern that is important for its function of suspending minerals and delivering the protein and minerals to the neonate in a readily ingestible form.
Acknowledgments The authors wish to thank Dr. Aladar Szalay for providing access to the fluorescence spectrophotometer in his laboratory for this study.
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