Secreted glycoprotein from Live Zaire ebolavirus-infected cultures: preparation, structural and biophysical characterization, and thermodynamic stability.
Milligram quantities of Zaire ebolavirus nonstructural, secreted glycoprotein (sGP) were purified to homogeneity, and this preparation was characterized by an array of biophysical and biochemical experiments. Mass-spectrometry analysis revealed sGP posttranslational modifications and regions susceptible to limited proteolysis. In solution, sGP has an absolute molar mass of 103 kDa, is monodisperse, and folds into a predominantly beta -sheet conformation with a distinct tertiary structure. sGP appears to have a unique free-energy landscape that facilitates reversible folding and a strong propensity for disulfide-linked dimeric quaternary structure under a wide range of conditions; the low apparent free energy of conformation transition of sGP ( Delta G=1.7+/-0.1 kcal/mol) suggests that the molecule is well suited as a thermodynamically facile switch, which would allow it to report on relatively subtle changes in milieu. In addition, a conformational transition at 37 degrees C was detected in thermal denaturing experiments. On the basis of biophysical and biochemical considerations alone, we propose that the property of being a thermodynamically facile switch is an important clue to reveal sGP functionality.
Barrientos
Laura G
LG
Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. LBarrientos1@cdc.gov
Martin
Amy M
AM
Wohlhueter
Robert M
RM
Rollin
Pierre E
PE
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002522:Chlorocebus aethiops; D015023:Democratic Republic of the Congo; D004355:Drug Stability; D029043:Ebolavirus / Q000302:isolation & purification / Q000502:physiology*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D000882:Haplorhini; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D006023:Glycoproteins; D014764:Viral Proteins