By altering the building blocks of brazzein, a natural protein far sweeter than sugar, University of Wisconsin-Madison scientists found a couple of changes that actually made it sweeter.

Six proteins in nature taste sweet to people. Scientists know the structure of three – brazzein, monellin and thaumatin, according to John Markley, who directed the study. These sweet proteins show how unexpected nature”s chemistry can be.

“Nature has produced several sweet-tasting proteins and the ones whose shapes we know have dissimilar structures and DNA sequences,” says Markley. In fact each of the sweet proteins from plants appears to have evolved from different proteins with completely different roles.

As scientists rush to discover the roles of proteins that are written in plant, animal and human genes, they often look for clues to a protein”s function from its amino acid sequence. Proteins with similar sequences often have similar functions, according to Markley. In the absence of sequence similarities, the protein”s structure – its shape – can be helpful. Proteins that fold up into similar structures often have similar functions even though their sequences may be quite different.

“On the basis of brazzein”s sequence and even structure, we never would have predicted that it would taste sweet,” Markley says. “Brazzein”s sequence suggests it is similar to a plant protein that interferes with insect digestion. Its fold has similarities to that of a toxin in scorpion venom! Our research shows how important it is to study proteins in their biological context.”

Markley, a structural biologist who directs the Nuclear Magnetic Resonance Facility at the UW-Madison, along with protein chemist Fariba Assadi-Porter and microbiologist David Aceti, reported their findings in the Archives of Biochemistry and Biophysics. The three are with the College of Agricultural and Life Sciences.

Goran Hellekant, a neurophysiologist in the UW-Madison School of Veterinary Medicine, first purified and identified brazzein from the fruit of a West African plant. Sweet fruits are thought beneficial to plants because they attract animals that disperse plant seeds to new locations where they can take root. Markley and Hellekant led a team that described brazzein”s structure in 1998.

Brazzein”s natural origin and its stability over a range of temperatures and acidic conditions make it an ideal candidate for a low-calorie sweetener, Markley notes.

Brazzein is the smallest known sweet-tasting protein, he says. Its small size makes it an ideal molecule for investigating what it is about the chemical and structural nature of these proteins that results in their sweet taste.

The African fruit contains two forms of brazzein, Markley says. The main form has 54 amino acids and is 9,500 times sweeter than sugar on a per molecule basis. A minor form has an identical structure but is missing a terminal amino acid; it is twice as sweet as the main form.

Markley, Assadi-Porter and Aceti developed methods to produce 15 brazzein-like proteins – each differing from brazzein by one amino acid change at a different location along the sequence. Since altering the protein”s shape should affect sweetness, the researchers used Nuclear Magnetic Resonance techniques to verify that each of the 15 proteins adopted the same overall fold as brazzein. One of the 15 altered proteins failed to fold properly and was excluded from the study.

“Folding creates a structural scaffold,” Markley explains. “The other 14 had the same shape as brazzein.”

A taste panel compared these 14 brazzein-like proteins to sugar and to brazzein from fruit. Four of the 14 compounds exhibited almost no sweetness, six had significantly reduced sweetness, two were as sweet as the sweeter form of brazzein in fruit and two were twice as sweet as that compound.

“We believe that although all 14 had the same shape as brazzein, the amino acid alterations created differences in the side chains that affected sweetness,” Markley says.

The study found that there are two regions of the molecule that are particularly important for sweetness. “Changes in those regions altered the sweetness intensity,” Markley says. “We propose that those regions are involved in binding to the receptor.”

The researchers want to know how brazzein fits into the receptor to trigger the sweet taste it elicits in people and monkeys. The receptor for sweet taste has not yet been isolated. Markley hopes structural features of brazzein will help scientists find the receptor to which it binds.

The research was supported by: state funding to the UW-Madison College of Agricultural and Life Sciences, and grants from the National Institutes of Health (NIH) and the UW-Madison Graduate School. The nuclear magnetic resonance studies were supported by the NIH Biomedical Technology Program, with additional equipment funding from UW-Madison, the National Science Foundation, the NIH and U.S. Department of Agriculture.