Determination of molecular weight of the protein moiety in protein-detergent complexes without direct knowledge of detergent binding.

Abstract

Sedimentation equilibrium measurements can be used to determine the molecular weight of the protein moiety of a protein-detergent complex without prior knowledge of detergent binding. The procedure is to adjust the solvent density by addition of D20 so as to blank out the contribution of bound detergent to the sedimentation potential. An approximate measure of detergent binding can be obtained from the effect of solvent density on the sedimentation result. The procedure is also applicable to protein-lipid complexes. It The purpose of this paper is to show that the difficulty created by inability to determine the amount of bound detergent can be circumvented by using D20 to adjust the density of the solvent to the point where it matches the buoyant density of the detergent. The detergent then makes no contribution to the sedimentation potential, no matter how much is bound. partial specific volume. Their purpose was different from ours, but the underlying physical principle is the same. D20 has been used to blank out the detergent contribution in neutron scattering by protein-detergent complexes (5), and sucrose has been used for the same purpose in x-ray scattering (6).THEORETICAL BASIS Treatment of sedimentation equilibrium in terms of the thermodynamics of multicomponent solutions (7)

Structure of an HIV-1–neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer

“…Prolines and N-terminal resonances in the foldon domain that could not be observed due to deficiencies in spectral quality were omitted. remove the contribution of the detergent to the observed buoyant molecular weights (48,49). The equilibrium association constant was estimated by globally fitting the data to a three-step binding model using the software Heteroanalysis v. 1.1.44 (J. L. Cole and J. W. Lary, University of Connecticut).…”

Oligomeric State of the Colon Carcinoma-associated Glycoprotein GA733-2 (Ep-CAM/EGP40) and Its Role in GA733-mediated Homotypic Cell-Cell Adhesion

“…Fractions containing purified GA733-FL were then concentrated to 0.1-0.4 mg/ml and dialyzed for at least 24 h at 4°C against PBS containing 0.15 mM PMSF and 0.5 mM C 12 E 8 in 22% D 2 O (v/v). In the presence of detergent, the protein is part of a proteindetergent complex that has a buoyant molecular mass, M c (1 Ϫ Ј), containing contributions from the protein and the bound detergent (35,36),…”

Active Detergent-solubilized H+,K+-ATPase Is a Monomer

“…Previously, introducing density variation by exchange with heavy water has been established in sedimentation equilibrium experiments as a useful method to estimate the contribution that the partial specific volumes makes to M b , the buoyant molar mass of the solubilized complex (59). In appropriate cases, this method can be used, by detergent density matching, to blank out the effect of detergent from the buoyancy term in sedimentation equilibrium experiments of membrane proteins (60). Recently, D 2 O exchange has also been used to extend the scope for c(s) sedimentation velocity analysis, as a means to unravel the hydrodynamic properties of samples that are not homogeneous or stable (61,62), and to account for the complexity of solubilized membrane proteins (63).…”