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J. Biol. Chem., Vol. 278, Issue 33, 31331-31339, August 15, 2003

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Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy^*

Fariba M. Assadi-Porter , Frits Abildgaard, Heike Blad and John L. Markley

From the National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (transhydrogen bond couplings). We have applied this approach to screen a series of five single site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long range, three-dimensional data correlating connectivities among backbone ¹H^N, ¹⁵N, and ¹³C' atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three "sweet" variants exhibited the same pattern of hydrogen bonds, whereas all three "non-sweet" variants lacked one hydrogen bond at the middle of the -helix, where it is kinked, and one hydrogen bond in the middle of -strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.

Received for publication, March 17, 2003 , and in revised form, April 30, 2003.

NMR chemical shifts and trans-hydrogen bond J couplings have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under acquisition codes 5723 (wild-type brazzein), 5725 (Ala² insertion), 5727 (H31A), 5728 (R33A), 5724 (R43A), and 5726 (D50A).

^* This work was supported by National Institutes of Health Grant RR 02301 (to J. L. M.) and by an Applied Research Grant (to F. M. A.-P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: National Magnetic Resonance Facility, Dept. of Biochemistry, University of Wisconsin, Madison, WI 53706. Tel.: 608-262-4687; Fax: 608-262-3759; E-mail: fariba{at}nmrfam.wisc.edu.

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