Comparison of Limulus alpha-macroglobulin with human alpha2-macroglobulin: thiol ester characterization, subunit organization, and conformational change.
The properties of the thiol ester-containing alpha-macroglobulin (alphaM) from the horseshoe crab (Limulus polyphemus) have been compared with those of the human analogue (alpha2M). Thiol ester accessibility was more restricted in Limulus alphaM than in human alpha2M. Fluorescent probes attached to the thiol ester cysteine indicated very similar local environments in the cleaved state of the two alphaMs. The separation between the two thiol ester cysteines in the cleaved state, determined by fluorescence resonance energy transfer, was also very similar for the two alphaMs. Differences were found in the oligomerization state and conformational changes of the two proteins. Whereas human alpha2M appears to be exclusively a dimer of dimers, Limulus alphaM can exist in both tetrameric and dimeric forms, although with marked preference for the dimer. Conformational change within a dimeric trapping unit, monitored by 6-(p-toluidino)-2-napthalene-sulfonic acid fluorescence change, showed that each monomer of the Limulus alphaM dimer appears to change conformation independently, whereas human alpha2M requires both thiol esters within a functional unit to be cleaved before the conformational change occurs. Taken together, these findings indicate that, whereas individual thiol esters in both types of alphaM are similar in properties, differences in subunit-subunit interaction result both in differences in state of oligomerization and in cooperativity of conformational change, which may reflect a fundamentally different organization of the subunits within a dimer in the two alphaMs.
Bowen
M E
ME
Department of Biochemistry, University of Illinois-Chicago, 60612-4316, USA.
Armstrong
P B
PB
Quigley
J P
JP
Gettins
P G
PG
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004952:Esters / Q000737:chemistry; D005456:Fluorescent Dyes; D006737:Horseshoe Crabs / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000737:chemistry*
D004952:Esters; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D003545:Cysteine