Presence Of Folding Intermediates Research Articles

Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions.

Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under "gentle" (i.e., low energy) ion transmission conditions with structures measured under "harsh" (i.e., higher energy) transmission. This work confirms that when using "gentle" instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, "harsh" conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.

The Skp Chaperone Helps Fold Soluble Proteinsin Vitroby Inhibiting Aggregation

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.

Common Structural Stability Properties of 4-Helical Bundle Cytokines: Possible Physiological and Pharmaceutical Consequences

Biological activity and clinical efficacy of a therapeutic protein are contingent upon the structural stability, bioavailability, and clearance rates of the protein. In this review, we examine the class of 4-helical bundle cytokines for common stability properties that may affect biological structure and efficacy. Three critical stability features that are hallmarks of this class of cytokines are the pH dependence of structural stability, the presence of folding intermediates, and the population of aggregation intermediates. We hypothesize that certain cytokines have increased stability in acid to enable receptor-mediated clearance, and that reengineering local endocytic trafficking can result in dramatic improvements in global serum half-life and therapeutic efficacy. The common feature of folding and aggregation intermediates has implications on kinetic folding pathways, membrane permeability, solubility, and precipitation properties that are critical for commercial production, formulation, and delivery. Understanding the structural stability properties of this class of cytokines may help elucidate new approaches to improving therapeutic efficacy.

Folding and conformational studies on SCR1-3 domains of human complement receptor 1.

Short consensus repeats SCR3 and SCR1-3 are soluble recombinant proteins, consisting of the third and first three N-terminal domains of complement receptor 1, respectively, which retain some anti-complement activity. The conformational stabilities and folding/unfolding of SCR3 and SCR1-3 have been studied using circular dichroism and equilibrium and pre-equilibrium fluorescence spectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl) is rapid and completely reversible. Reduction of disulphide bridges in the folded proteins by beta-mercaptoethanol leads to an increase in fluorescence intensity. The fluorescence intensity of the folded proteins is approximately 7.5% of that of the respective unfolded proteins. The data can be approximated to a two-state transition between native and denatured forms of the proteins. SCR3 has a conformational stability in water of 12-13 kJ/mol whereas that of SCR1-3 is 19.5-19.9 kJ/mol depending upon the technique utilized. The heat capacity change associated with the unfolding of SCR1-3 was obtained by a series of GdnHCl unfolding experiments over a range of temperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol(residue). The refolding process of SCR3 was found to be simple, described by a single exponential equation, whereas that of SCR1-3 was found to be complex and could be fitted to a double exponential equation indicating the presence of folding intermediates.

Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates

The trans → cis isomerization of Pro 93 was measured during refolding of bovine ribonuclease A. This isomerization is slow (τ = 500s) under marginally stable folding conditions of 2.0 M GdmCl, pH 6, at 10°C. However, it is strongly accelerated (τ = 100 s) in samples which, prior to isomerization, had been converted to a folding intermediate by a 15s refolding pulse under strongly native conditions (0.8 M ammonium sulfate, 0°C). The results demonstrate that extensive folding is possible before Pro 93 isomerizes to its native cis state and that the presence of structural folding intermediates leads to a marked increase in the rate of subsequent proline isomerization.