Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin.
Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.
De Lorenzi
Ersilia
E
Department of Pharmaceutical Chemistry, University of Pavia, Viale Taramelli 12, I-27100 Pavia, Italy. ersidelo@unipv.it
Grossi
Silvia
S
Massolini
Gabriella
G
Giorgetti
Sofia
S
Mangione
Palma
P
Andreola
Alessia
A
Chiti
Fabrizio
F
Bellotti
Vittorio
V
Caccialanza
Gabriele
G
D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D006639:Histidine; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms; D013498:Suramin / Q000737:chemistry*; D014443:Tyrosine; D001613:beta 2-Microglobulin / Q000737:chemistry*
D020033:Protein Isoforms; D001613:beta 2-Microglobulin; D014443:Tyrosine; D006639:Histidine; D013498:Suramin; D003300:Copper