The effect of conserved residue charge reversal on the folding of recombinant non-phosphorylated human beta-casein.
A short stretch of 13 amino acids in the central portion of human beta-casein contains four positively charged conserved residues, three Lys and one Arg. We changed these individually to Glu, reversing their charge, and compared the resulting recombinant proteins to the wild-type recombinant, monitoring thermal aggregation with turbidity as well as using the fluorescence of the intrinsic Trp, of hydrophobically bound ANS and fluorescence resonance energy transfer from Trp to ANS to detect differences in structure. The results demonstrate the need to maintain the actual or functional identity of these conserved charged amino acid residues in order to attain the protein folding and functional properties of the wild-type human beta-casein molecule. They emphasize the probability that native human beta-casein has a unique folding pattern that is important for its function of suspending minerals and delivering the protein and minerals to the neonate in a readily ingestible form.
Department of Biochemistry and Microbiology, Biochemistry Division, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
Hongyin
H
Bu
Satish M
SM
Sood
Charles W
CW
Slattery
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates; D002364:Caseins / Q000737:chemistry* / Q000145:classification*; D017124:Conserved Sequence; D031541:Fluorescence Resonance Energy Transfer; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009391:Nephelometry and Turbidimetry; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D000596:Amino Acids; D000817:Anilino Naphthalenesulfonates; D002364:Caseins; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan