Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins

Abstract

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The L L-strand-rich single chain Fv fragment (scFv) and K K-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is K K-helix rich, are predominated by K K-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the K K-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with nativelike structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.

Sequence determinants of protein aggregation: tools to increase protein solubility

“…First, most amyloid fibrils are SDS-insoluble, whereas SDS can usually dissolve IBs. This observation is in agreement with the higher extent of β-sheet content of amyloids relative to that in IBs, in which the presence of some native or disordered structure can be still detected [27,60]. As a result amyloids would display more and stronger intermolecular non-covalent interactions that would provide them with higher order and stability in front of denaturation, while sharing similar overall connectivity between polypeptide chains than this present in IBs.…”

Generation of catalytic protein particles in Escherichia coli cells using the cellulose-binding domain from Cellulomonas fimi as a fusion partner

“…4A). Next, the FTIR was used to analyze the β-sheet and α-helix composition in the protein particles [24,25]. The GusA-CBD and BglA-CBD particle spectral peaks were separated using a peak deconvolution program (Cutter 5.0), and the spectra were divided into discrete β-sheet and α-helix profiles (Fig.…”

Kinetics of inclusion body formation studied in intact cells by FT‐IR spectroscopy

“…Indeed, the second derivative analysis of the protein Amide I band [10][11][12] allows determining the protein secondary structure, since Amide I is a structure sensitive band, being mainly due to the absorption of the carbonyl C‚O group of the peptide bond. Interestingly, it has been shown that the analysis of the Amide I band components enables also the detection of protein aggregates [4,6,7,13], which are not easily monitored by other optical spectroscopies.…”