Probing the sweet determinants of brazzein: Wild-type brazzein and a tasteless variant, brazzein-ins(R 18a -I 18b ), exhibit different pH-dependent NMR chemical shifts q Qin Zhao a, * , Jikui Song a , Zheyuan Jin b , Vicktoria Danilova b , Go ¨ ran Hellekant b , John L. Markley a a Department of Biochemistry, University of Wisconsin–Madison, College of Agricultural and Life Sciences, USA b Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, USA Received 27 June 2005 Available online 27 July 2005 Abstract Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg- Ile) were inserted between Leu 18 and Ala 19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single a-helix, and around the Cys 16 –Cys 37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the a-helix and the second b-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His 31 , Arg 33 , Glu 41 , Arg 43 , Asp 50 , and Tyr 54 ) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His 31 , Glu 41 , Asp 50 , and Tyr 54 were unal- tered by the insertion. The insertion led to large chemical shift and pK a perturbation of Glu 36 , a residue shown previously to be important for brazzeinÕs sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further sup- port to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been pos- tulated for brazzein and other sweet proteins (monellin and thaumatin). Ó 2005 Elsevier Inc. All rights reserved. Keywords: Brazzein; Sweet-taste determinants; NMR spectroscopy; pK a ; Mutagenesis Seven proteins from different plants have been found to induce or modify sweet taste in humans and Old World primates. Their primary amino acid sequences and tertiary structures differ from one another ([1,2] and references therein), and all have been found to be- long to protein families that include members with other known functions. Thus, the sweet properties of each protein appear to have evolved separately. (1) Monellin from Dioscoreophyllum cumminsii (West Africa) is struc- turally related to cystatin [3–5]. (2) Thaumatin from Thaumatococcus danielli (West Africa) resembles plant defense proteins; one of its homologues can bind (1,3)- b-D-glucan [6]. (3) Mabinlin from Capparis masaikai (subtropical Yunnan, China) has sequence homology to seed storage proteins from several plants [7,8]. (4) Brazzein from Pentadiplandra brazzeana (Africa) has se- quence and three-dimensional structural similarity to ra- peseed serine proteinase inhibitors and a group of plant defensins [1,2,9]; pentadin, which is isolated from a smoke-dried form of the same fruit, probably is simply a dimeric form of brazzein [1]. (5) Curculin from Curcul- igo latifolia (Malaysia) is also a non-functional 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.049 q This work was supported by NIH Grants DC006016 (G.H.) and RR02301 (J.L.M.). Chemical shifts and pK a values described here have been deposited at BioMagResBank (BMRB 5295 for brazzein and BMRB 5296 for brazzein-ins(R 18a -I 18b )). * Corresponding author. Fax: +1 608 262 3759. E-mail address: qin@nmrfam.wisc.edu (Q. Zhao). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 335 (2005) 256–263 BBRC