Cyclic peptide models of the Ca2+-binding loop of alpha-lactalbumin.
A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca2+-binding loop of alpha-lactalbumin (LA). All amino acids of the Ca2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys79-Phe-Leu-Asp82-Asp-Asp-Leu-Thr- Asp87-Asp88, where three carboxylates of Asp82, Asp87, and Asp88 and the amide carbonyl oxygen atoms of Lys79 and Asp84 participate in Ca2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87-88) and nonbinding Asp residues (83-84) in coordinating the cation. The structural features of synthetic peptides and their Ca2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca2+ was dependent on the structure and concentration of the model and the Ca2+ to peptide ratio (r(cat)). A surprising time dependence of the FTIR spectra of Ca2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after approximately 60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca2+ took more time. Infrared spectra showed that in the Ca2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO- groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca2+-binding system.
Farkas
Viktor
V
Department of Organic Chemistry, Eötvös Loránd University, PO Box 32, H-1518 Budapest 112, Hungary.
Vass
Elemér
E
Hanssens
Ignace
I
Majer
Zsuzsa
Z
Hollósi
Miklós
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared
D010456:Peptides, Cyclic; D007768:Lactalbumin; D002118:Calcium