Colloid and Polymer Science

, Volume 274, Issue 2, pp 117–137 | Cite as

Comparative studies of structural properties and conformational changes of proteins by analytical ultracentrifugation and other techniques

  • H. Durchschlag
  • P. Zipper
  • G. Purr
  • R. Jaenicke
Original Contribution

Abstract

Analytical ultracentrifugation is a powerful tool for investigating the size of proteins in solution, especially by measuring sedimentation and diffusion coefficients and molar masses. Several further molecular parameters such as frictional ratios, axial ratios of hydrodynamic models, and Stokes radii allow a rough estimate of the protein overall structure. Sedimentation analysis may also be applied efficaciously for monitoring conformational changes of proteins occurring upon ligand binding or denaturation. For the determination of very small changes in shape, however, great care and a series of precautions are required. We investigated the enzymes citrate synthase and malate synthase in the absence and in the presence of ligands, in order to study the structural properties of the proteins and their ligand complexes. We also compared the results of the ultracentrifugal analysis with the results of other solution techniques such as UV absorption, fluorescence spectroscopy, circular dichroism, and small-angle x-ray scattering on the one hand, and the crystallographic 3D structure of citrate synthase on the other. The spectroscopic methods may be used as efficient and rapid tools for screening the occurrence of conformational changes caused by alterations of chromophores and fluorophores. The structural information provided by small-angle scattering (e.g., radii of gyration, maximum particle diameters, vclumes and surface areas) can be used to establish quantitative correlations between solution scattering and hydrodynamic data. In this context, however, knowledge or qualified assumptions of partial specific volumes and hydration are additionally required. Good agreement was reached between small-angle scattering and ultracentrifugal data, and also with crystallographic data if protein hydration was considered properly. The given approaches may be used to predict hydrodynamic properties if x-ray data are available, and for many verifications of other structural data, e.g., Stokes radii, diffusion coefficients, axial and frictional ratios determined by independent methods.

Key words

Proteins citrate synthase malate synthase analytical ultracentrifugation small-angle scattering comparative studies predictions structural properties hydrodynamic modeling conformational changes 

Abbreviations materials

AcCoA

acetyl coenzyme A

CoA

coenzyme A

CS

citrate synthase (EC 4.1.3.7)

DTT

dithiothreitol

GdrnCl

guanidinium chloride

MS

malate synthase (EC 4.1.3.2.)Methods

AUC

analytical ultracentrifugation

CD

circular dichroism

EM

fluorescence emission spectroscopy

EX

fluorescence excitation spectroscopy

SAS

small-angle scattering

SAXS

small-angle x-ray scattering

UV

ultraviolet absorption spectroscopy

XD

x-ray diffraction

Models

OE

oblate ellipsoidal model

PE

prolate ellipsoidal model

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Copyright information

© Steinkopff Verlag 1996

Authors and Affiliations

  • H. Durchschlag
    • 1
  • P. Zipper
    • 2
  • G. Purr
    • 1
  • R. Jaenicke
    • 1
  1. 1.Institute of Biophysics and Physical BiochemistryUniversity of RegensburgRegensburgGermany
  2. 2.Institute of Physical ChemistryUniversity of GrazGrazAustria

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