Format

Send to

Choose Destination
Methods Enzymol. 2015;562:187-204. doi: 10.1016/bs.mie.2015.04.004. Epub 2015 Jun 19.

Elucidating Complicated Assembling Systems in Biology Using Size-and-Shape Analysis of Sedimentation Velocity Data.

Author information

1
Graduate Program in Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
2
Division of Immunobiology and Center for Systems Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA; Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA. Electronic address: andrew.herr@cchmc.org.

Abstract

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has seen a resurgence in popularity as a technique for characterizing macromolecules and complexes in solution. SV-AUC is a particularly powerful tool for studying protein conformation, complex stoichiometry, and interacting systems in general. Deconvoluting velocity data to determine a sedimentation coefficient distribution c(s) allows for the study of either individual proteins or multicomponent mixtures. The standard c(s) approach estimates molar masses of the sedimenting species based on determination of the frictional ratio (f/f0) from boundary shapes. The frictional ratio in this case is a weight-averaged parameter, which can lead to distortion of mass estimates and loss of information when attempting to analyze mixtures of macromolecules with different shapes. A two-dimensional extension of the c(s) analysis approach provides size-and-shape distributions that describe the data in terms of a sedimentation coefficient and frictional ratio grid. This allows for better resolution of species with very distinct shapes that may co-sediment and provides better molar mass determinations for multicomponent mixtures. An example case is illustrated using globular and nonglobular proteins of different masses with nearly identical sedimentation coefficients that could only be resolved using the size-and-shape distribution. Other applications of this analytical approach to complex biological systems are presented, focusing on proteins involved in the innate immune response to cytosolic microbial DNA.

KEYWORDS:

Analytical ultracentrifugation; Frictional ratio; Sedimentation coefficient; Sedimentation velocity; Size-and-shape analysis

PMID:
26412652
PMCID:
PMC5765866
DOI:
10.1016/bs.mie.2015.04.004
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center