Destabilization of pea lectin by substitution of a single amino acid in a surface loop

Abstract

Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability of Pisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val103) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (Tm) by some 10 degrees C in comparison with wild-type (wt) PSL, and the denaturation energy (delta H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe104) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugar-binding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced in Escherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 degrees C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are active in planta at ambient temperatures, but are inactive and possibly non-toxic at 37 degrees C in the intestines of mammals.

A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b5

“…Loops are known to affect the stability of a protein fold (Gekko et al, 1993;Hoedemaeker et al, 1993;Dion-Schultz & Howell, 1997;Fetrow et al, 1997). This is illustrated notably by circularly permuted proteins (Garrett et al, 1996;Uversky et al, 1996) where natural turns and artificial linkers can make the difference between a well-folded protein and a molten globule.…”

Omega loops; nonregular secondary structures significant in protein function and stability

“…Thus far, fl-loops have always been found at the protein surface and their lack of regular hydrogen bonding patterns suggests that these structures do not play a role in protein structure and stability, however, single and multiple mutations in some fl-loops can have drastic effects on overall protein stability. A single change from a valine to an alanme in an fl-loop in pea lectin caused a 100 decrease in the melting temperature of the protein (42). The researchers suggest this destabilization is caused by a loss of hydrophobic contacts between the loop and the protein.…”

Correct Post‐Translational Modification and Stable Vacuolar Accumulation of Phytohemagglutinin Engineered to Contain Multiple Methionine Residues

“…Using this approach, we have shown that mutants of PHA containing three and four methionine residues stably accumulate in the vacuoles of tissue-cultured cells and the protein storage vacuoles of seeds. Although minor modifications of seed storage proteins have been shown not to affect transport and targeting, one must be cautious with this approach since a report by Hoedemaeker et al (1993) showed that pea lectin was thermally destabilized by the modification of a single amino acid. Recently, Dyer et al (1993) proposed a similar method to replace individual amino acids with methionine residues for the seed storage protein phaseolin although the replacements were based on conserved hydrophobic residues, not conserved methionine residues.…”