Studying Protein Folding and Aggregation by Laser Light Scattering

Abstract

Originally published in: Protein Folding Handbook. Part I. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30784‐2 The sections in this article are Introduction Basic Principles of Laser Light Scattering Light Scattering by Macromolecular Solutions Molecular Parameters Obtained from Static Light Scattering (SLS) Molecular Parameters Obtained from Dynamic Light Scattering (DLS) Advantages of Combined SLS and DLS Experiments Laser Light Scattering of Proteins in Different Conformational States – Equilibrium Folding/Unfolding Transitions General Considerations, Hydrodynamic Dimensions in the Natively Folded State Changes in the Hydrodynamic Dimensions during Heat‐induced Unfolding Changes in the Hydrodynamic Dimensions upon Cold Denaturation Denaturant‐induced Changes of the Hydrodynamic Dimensions Acid‐induced Changes of the Hydrodynamic Dimensions Dimensions in Partially Folded States – Molten Globules and States Comparison of the Dimensions of Proteins in Different Conformational States Scaling Laws for the Native and Highly Unfolded States, Hydrodynamic Modeling Studying Folding Kinetics by Laser Light Scattering General Considerations, Attainable Time Regions Hydrodynamic Dimensions of the Kinetic Molten Globule of Bovine α‐Lactalbumin RNase A is Only Weakly Collapsed During the Burst Phase of Folding Misfolding and Aggregation Studied by Laser Light Scattering Overview: Some Typical Light Scattering Studies of Protein Aggregation Studying Misfolding and Amyloid Formation by Laser Light Scattering Overview: Initial States, Critical Oligomers, Protofibrils, Fibrils Aggregation Kinetics of Aβ Peptides Kinetics of Oligomer and Fibril Formation of PGK and Recombinant Hamster Prion Protein Mechanisms of Misfolding and Misassembly, Some General Remarks Protocols and Instrumentation Laser Light Scattering Instrumentation Basic Experimental Set‐up, General Requirements Supplementary Measurements and Useful Options Commercially Available Light Scattering Instrumentation Experimental Protocols for the Determination of Molecular Mass and Stokes Radius of a Protein in a Particular Conformational State Acknowledgments

Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing

“…It has been well established that the hydrodynamic dimension of a protein increases upon denaturation, and thus DLS can be used to characterize protein-folding states. 10 In this study, we demonstrated that the r H value measured by DLS provides enough resolution to capture multiple intermediate states of BSA upon denaturation. There are several advantages of using DLS to monitor protein-folding states over spectroscopy-based methods such as CD and fluorescence.…”

“…DLS is well known for the quick, nonperturbing nature of measurement, and for the ability to measure a wide spectrum of particles ranging from 1 nm to 5 μm. In addition, as the hydrodynamic dimension of a protein increases upon denaturation, DLS has also been successfully applied in studying protein‐folding states …”

“…DLS is well known for the quick, nonperturbing nature of measurement, and for the ability to measure a wide spectrum of particles ranging from 1 nm to 5 :m. In addition, as the hydrodynamic dimension of a protein increases upon denaturation, DLS has also been successfully applied in studying protein-folding states. 9,10 In this work, we sought to develop a DLS-based method for the in-process monitoring of IB solubilization, protein refolding, and aggregation near the production line. We first demonstrated the reliability of DLS in monitoring protein unfolding using bovine serum albumin (BSA).…”

ATP‐dependent modulation and autophosphorylation of rapeseed 2‐Cys peroxiredoxin

“…Given the essential role of ATP in the peroxidase activity, we evaluated changes in the structure of 2-Cys Prx brought about by the concerted action of the nucleotide and the bivalent cation. To accurately determine the molecular mass of our 2-Cys Prx preparations, static light-scattering measurements were performed, because this spectroscopic technique allows direct estimation of the species in solution [23]. In the absence of perturbants, the predominant form of After the addition of EGTA to a final concentration of 5 mM, the protein solution was further incubated for 5 min and the peroxidase activity was assayed as in (A).…”

Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins

“…A key motivation is that many proteins are marginally stable and only fold in the presence of their ligands or binding partners, opening new regulatory possibilities (1,2). An important reason for recent progress is the growing availability of methods that provide structural information on these conformationally heterogeneous systems, such as NMR (3), scattering methods (4,5), and single-molecule FRET (6)(7)(8). Although NMR provides mostly local details, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and single-molecule FRET provide overall hydrodynamic or long-range distance information.…”