As a follow-up to our recent analysis of the electrostatics of bovine beta-lactoglobulin (Eberini et al. in Amino Acids 42:2019-2030, 2011), we investigated whether the occurrence in the native structure of calycins-the superfamily to which beta-lactoglobulin belongs-of amino acids with anomalous pK(a)s is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK(a) of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the 'normalization' of anomalous pK(a)s may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.

Wards in the keyway: amino acids with anomalous pKas in calycins / I. Eberini, C. Sensi, M. Bovi, H. Molinari, M. Galliano, F. Bonomi, S. Iametti, E. Gianazza. - In: AMINO ACIDS. - ISSN 0939-4451. - 43:6(2012 Dec), pp. 2457-2468. [10.1007/s00726-012-1324-9]

Wards in the keyway: amino acids with anomalous pKas in calycins

I. Eberini
Primo
;
C. Sensi
Secondo
;
F. Bonomi;S. Iametti
Penultimo
;
E. Gianazza
2012

Abstract

As a follow-up to our recent analysis of the electrostatics of bovine beta-lactoglobulin (Eberini et al. in Amino Acids 42:2019-2030, 2011), we investigated whether the occurrence in the native structure of calycins-the superfamily to which beta-lactoglobulin belongs-of amino acids with anomalous pK(a)s is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK(a) of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the 'normalization' of anomalous pK(a)s may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.
Calycins; Electrostatics; Unfolding; Urea; Electrophoresis; Molecular modeling
Settore BIO/10 - Biochimica
Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica
Settore CHIM/06 - Chimica Organica
dic-2012
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/196258
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