Denaturant-induced Expansion and Compaction of a Multi-domain Protein: IgG

Abstract

It is generally believed that unfolded or denatured proteins show random coil statistics and hence their radius of gyration simply scales with solvent quality (or denaturant concentration). Indeed, nearly all proteins studied thus far have been shown to undergo a gradual and continuous expansion with increasing denaturant concentration. Here, we use fluorescence correlation spectroscopy (FCS) to show that while protein A, a multidomain and predominantly helical protein, expands gradually and continuously with increasing concentration of guanidine hydrochloride (GdnHCl), the F(ab′) 2 fragment of goat anti-rabbit antibody IgG, a multi-subunit all β-sheet protein, however, does not show such continuous expansion behavior. Instead, it first expands and then contracts with increasing GdnHCl concentration. Even more striking is the fact that the hydrodynamic radius of the most expanded F(ab′) 2 ensemble, observed at 3-4 M GdnHCl, is ca. 3.6 times that of the native protein.Further FCS measurements involving urea and NaCl show that the unusually expanded F(ab′) 2 conformations might be due to electrostatic repulsions. Taken together, these results suggest that specific interactions need to be considered while assessing the conformational and statistical properties of unfolded proteins, particularly under conditions of low solvent quality. KeywordsFCS; Flory's random coil model; IgG; protein folding; unfolded state Despite its obvious importance in understanding how proteins fold, the unfolded or denatured state of proteins remains relatively unexplored. 1,2 Recent years have thus seen an increasing number of studies focused on the conformational properties of proteins in their unfolded state. 3-24 Of particular interest are those which assess the molecular dimensions as well as conformational dynamics of proteins under various denaturing conditions using ensemble 11-20 or single-molecule techniques. 25-32 All these studies, while involving a wide variety of peptides and proteins that span a large range of chain lengths (8-549 aa), generally support the notion that unfolded proteins behave as self-avoiding random-coils which undergo a continuous expansion with increasing denaturant concentration, following Flory's powerlaw relationship (i.e., R G = R 0 N ν ; where R G is the radius of gyration, R 0 is a constant determined *Corresponding author. Email address: gai@sas.upenn.edu. & L.G. and P.C. contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Figure 1a). Because of its distinctive structural characteristics and the prevalence of antibodies...

Unfolded-State Dynamics and Structure of Protein L Characterized by Simulation and Experiment

“…Trp-Cys quenching studies have shown that proteins L and G share similar denatured state structure and intramolecular dynamics20, with lower rates of intramolecular diffusion in decreasing amounts of denaturant. Similar findings have been made using single-molecule FRET21 and fluorescence correlation spectroscopy22.…”

How force unfolding differs from chemical denaturation

“…It is thus expected that the protein carries a positive charge of ∼+13e at pH 2 (SI Text), which could lead to mutual repulsion between residues and to a chain extension much less pronounced than that at neutral pH. Indeed, experiments suggest that the spatial extent of the unfolded state varies greatly with the nature of the denaturant and the presence of salts, highlighting the crucial role of repulsive electrostatic interactions (25).…”

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

“…The calculated values for hydrodynamic radii and intrinsic viscosities correlate to those reported for an intact antibody. [36][37][38] For instance, the reported hydrodynamic radius of intact IgG is 5.4 nm and the intrinsic viscosity is 6 ml/g. Altogether, these data show that the behavior of the newly designed oligospecific antibodies is as would be predicted from their domain structures ( Fig.…”