title: Crystallization and preliminary X-ray diffraction analysis of eukaryotic α2 -macroglobulin family members modified by methylamine, proteases and glycosidases.
abstract: α2 -Macroglobulin (α2 M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2 M (hα2 M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2 M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.
authors: Proteolysis Laboratory, Molecular Biology Institute of Barcelona, Barcelona, Spain.
authors: T
authors: T
authors: Goulas
authors: I
authors: I
authors: Garcia-Ferrer
authors: S
authors: S
authors: García-Piqué
authors: L
authors: L
authors: Sottrup-Jensen
authors: F X
authors: FX
authors: Gomis-Rüth
mesh_terms: D000818:Animals; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D008744:Methylamines / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors; D013820:Thermolysin; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D008744:Methylamines; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; C036828:ovomacroglobulin; C027451:methylamine; D006867:Hydrolases; D006026:Glycoside Hydrolases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D013820:Thermolysin
keywords: innate immunity; molecular oral microbiology