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Critical Regions for the Sweetness of Brazzein1 Provided by Elsevier - Publisher Connector FEBS 27205 FEBS Letters 544 (2003) 33^37 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Critical regions for the sweetness of brazzein1 provided by Elsevier - Publisher Connector Zheyuan Jina, Vicktoria Danilovaa, Fariba M. Assadi-Porterb, DavidJ. Aceti b, John L. Markleyb,Go«ran Hellekanta;Ã aDepartment of Animal Health and Biomedical Sciences, University of Wisconsin^Madison, Madison, WI 53706, USA bDepartment of Biochemistry, University of Wisconsin^Madison, Madison, WI 53706, USA Received25 February 2003; revised4 April 2003; accepted4 April 2003 First publishedonline 2 May 2003 Edited by Gianni Cesareni Brazzein is very water-soluble. Its isoelectric point (pI = 5.4) Abstract Brazzein is a small, heat-stable, intensely sweet pro- tein consisting of 54 amino acid residues. Based on the wild-type is lower than those of other sweet proteins, which all have brazzein, 25 brazzein mutants have been produced to identify pI s 7.0 [5]. Brazzein is remarkably heat-stable, andits sweet critical regions important for sweetness. To assess their sweet- taste remains after incubation at 80‡C for 4 h. Chemical mod- ness, psychophysical experiments were carried out with 14 hu- i¢cation studies suggested that the surface charge of the mol- man subjects. First, the results suggest that residues 29^33 and ecule is important andledto the conclusion that Arg, Lys, 39^43, plus residue 36 between these stretches, as well as the Tyr, His, Asp, andGlu are important for brazzein’s sweetness C-terminus are involved in the sweetness of brazzein. Second, andshouldbe studiedfurther [5,6]. In addition, studies with charge plays an important role in the interaction between braz- other sweet proteins, thaumatin andmonellin, have suggested zein and the sweet taste receptor. a role for lysine and/or carboxyl groups in the sweet protein^ ß 2003 Federation of European Biochemical Societies. Pub- receptor(s) interaction [7^9]. lished by Elsevier Science B.V. All rights reserved. The structure of brazzein was determined by 1H nuclear Key words: Brazzein; Sweet protein; Mutagenesis; magnetic resonance (NMR) spectroscopy in solution at pH Sweetness determinant; Sweetness evaluation; Taste 5.2 and22‡C [10]. The study revealed that brazzein contains one short K-helix (residues 21^29) and three strands of anti- parallel L-sheet (strand I, residues 5^7; strand II, residues 44^ 50; strand III, residues 34^39) held together by four disul¢de 1. Introduction bonds. The authors proposed that the small connecting loop containing His31 andthe randomcoil loop aroundArg43 Six sweet proteins have been discovered over the last 30 were the possible determinants of the molecule’s sweetness. years. The latest discovered is brazzein, isolated from the fruit Site-directed mutagenesis was used to change surface resi- of Pentadiplandra brazzeana Baillon [2]. Brazzein is a single- dues of WT brazzein at di¡erent locations along its sequence chain polypeptide of 54 amino acid residues with four intra- [11]; andtaste studiesof 14 brazzein variants showedthat molecular disul¢de bonds, no free sulfhydryl group, and no most mutations decreased sweetness but that two increased carbohydrate [3]. It is rich in lysine but contains no methio- sweetness. On the basis of these studies, the authors suggested nine, threonine or tryptophan. Brazzein exists in two forms in that the N- andC-termini and L-turn aroundArg43 are in- the ripe fruit. The major form contains pyroglutamate (pGlu) volvedin the sweetness of brazzein [1]. at its N-terminus; the minor form is without the N-terminal Here we present results from a larger set of brazzein var- pGlu (des-pGlu1). Taste comparisons of chemically synthe- iants, which includes multiple mutations at several speci¢c sizedbrazzein anddes-pGlu1brazzein revealedthat the latter positions, aimedat delineatingfurther how changes of charges protein has about twice the sweetness of the former [4].We andsidechains a¡ect the sweetness of brazzein. The ultimate use ‘WT (wild type) brazzein’ to denote recombinant des- goal, unrealizedas yet, is to predictsweetness from the chem- pGlu1 brazzein. ical structure of a compound, or from the amino acid se- quence of a sweet protein. Because New Worldprimates andother mammals donot perceive brazzein as sweet, inves- *Corresponding author. Fax: (1)-608-262 7420. tigations of its sweetness must be carriedout with humans or E-mail address: [email protected] (G. Hellekant). OldWorldprimates. This investigation describesandanalyzes 1 For brazzein mutants, the same nomenclature system was followed the e¡ects of site-directed mutations of brazzein on its sweet- as in the previous publication [Assadi-Porter et al., Arch. Biochem. ness as perceivedby human subjects. Biophys. 376 (2000) 259^265]. For example, Asp29Ala indicates the Asp of residue 29 was replaced by Ala; Arg19Ile20ins is a double insertion with Arg insertedat position 19 andIle insertedat 2. Materials and methods position 20; mutant Ala2Ala31 indicates the additive mutations of Ala2ins andHis31Ala. 2.1. Preparation of brazzein variants A protein expression system for WT brazzein, developed in Esche- Abbreviations: CNBr, cyanogen bromide; ddH2O, doubly deionized richia coli, allowedbrazzein variants to be producede⁄ciently [1]. water; MALDI, matrix-assistedlaser desorption/ionization; NMR, Brazzein mutants were preparedby site-directedmutagenesis(Quick nuclear magnetic resonance; pGlu, pyroglutamic acid; SCM, single- Change1 PCR kit, Stratagene) for substitutions of speci¢c amino chain monellin; SNase, staphylococcal nuclease; WT, wildtype; WT acids based on the template gene encoding WT and expressed as a brazzein, recombinant des-pGlu1 brazzein fusion protein (staphylococcal nuclease (SNase)^Met^brazzein). The 0014-5793 / 03 / $22.00 ß 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0014-5793(03)00383-1 FEBS 27205 21-5-03 Cyaan Magenta Geel Zwart 34 Z. Jin et al./FEBS Letters 544 (2003) 33^37 brazzein molecule was releasedfrom the fusion protein at the unique verbal labels: ‘barely detectable’, ‘weak’, ‘moderate’, ‘strong’, ‘very Met linkage by CNBr (cyanogen bromide) cleavage, then puri¢ed by strong’, and‘the strongest imaginable’. The intensity of the sweetness cation exchange chromatography, followedby reverse-phase high-per- was later convertedto a numerical value. The datawere averaged formance liquidchromatography puri¢cation to remove all unfolded between subjects, and standard deviations were calculated. Sweetness or mis-folded brazzein proteins [11]. scores were ¢rst evaluatedwith repeatedmeasurements ANOVA Because brazzein contains no tryptophan, its extinction coe⁄cient (analysis of variance) followedby pairwise comparisons of the scores was determined at 205 nm instead of 280 nm. The extinction coe⁄- for di¡erent variants using Fisher’s least signi¢cant di¡erences. Prob- cient O205 of each brazzein variant was calculatedfrom measurements ability P 9 0.05 was considered to be signi¢cant. of the absorbance of solutions at 280 and205 nm accordingto the O1mg=ml formula [12]: 205 = 27.0+120(A280/A205). All protein solutions were scannedat 195^290 nm at mediumspeed 3. Results with a Cary Win UV Scan spectrophotometer (Varian Analytical Instruments, Walnut Creek, CA, USA). Doubly deionized water 3.1. Characterization of brazzein variants (ddH2O) was usedas the blank, andthe baseline was subtracted 1 automatically. Samples were diluted by factors that provided absor- Fig. 1 shows the 1D H NMR spectra of two of the most bance values of about 0.7 at 205 nm; higher absorbances were sweet brazzein variants (Asp29Ala, Asp29Lys) andthree avoided to minimize e¡ects of stray light. The concentrations of braz- of the least sweet brazzein variants (Glu36Ala, Glu36Lys, zein solutions were determined from the absorbance at 205 nm and Glu36Gln). Each spectrum exhibits peaks at low frequency the extinction coe⁄cient for the particular variant. The molecular weights of the brazzein variants were con¢rmedwith (around0.4 ppm from methyl groups in the protein core) matrix-assistedlaser desorption/ionization (MALDI) mass spectrom- andat high frequency (around10.1 ppm from the hydroxyl etry. Samples were run on a Bruker Bi£ex III MALDI time-of-£ight proton of Tyr11) that are characteristic for folded protein. mass spectrometer in the linear mode using K-cyano-4-hydroxycin- Similar results obtainedfor the other mutants indicatedthat namic acidas the matrix. The external calibration was performed they too were correctly folded. with bovine insulin and ubiquitin as standards. One-dimensional (1D) 1H NMR spectroscopy was usedto check that the mutants were folded correctly. Each brazzein protein (0.5^1.0 3.2. Human evaluations 2 1 mM) was dissolved in 10% H2O at pH 7.0, and1D H NMR spectra Fig. 2 shows the average sweetness scores of all stimuli were acquiredwith a Bruker DMX 500 MHz spectrometer in a 5 mm used. In Fig. 2 we use black columns to indicate signi¢cantly 1H probe at 298 K. increasedsweetness, gray columns for no change, striped 2.2. Stimuli and sensory analysis columns for signi¢cantly less sweet than WT brazzein, and WT brazzein, brazzein variants, monellin, andsingle-chain monellin white columns for no di¡erence from water. The bars denote (SCM) were dissolved in ddH2O. The solutions were adjusted to pH S.E.M. 7.0 with 0.1 M NaOH or HCl. The concentrations of monellin and Four brazzein mutants (Asp29Ala, Asp29Lys, Asp29Asn, SCM were determined spectrophotometrically using a calculated mo- 31 31 andGlu41Lys) were scoredsigni¢cantly sweeter than WT lar extinction coe⁄cient O280 of 15 930 M cm [13] anda mass of 11 197 Da for monellin and11 050 Da for SCM, respectively (SWISS- brazzein. Three brazzein mutants (Ala2ins, Asp2Asn, and PROT). Gln17Ala) were scoredas sweet as WT brazzein. In eight The taste panel consistedof six females andeight males (ages 17^70 mutants the sweetness decreased signi¢cantly but the score years) of reportedgoodhealth andnormal sense of taste. The exper- imental protocol was approvedby the University of Wisconsin^Mad- was di¡erent from that of water, and in 10 mutants the sweet- ison Human Subjects Committee.
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