Next Article in Journal
Biomarkers for Predicting Clinical Outcomes of Chemoradiation-Based Bladder Preservation Therapy for Muscle-Invasive Bladder Cancer
Previous Article in Journal
Proteomics and Lipidomics in Inflammatory Bowel Disease Research: From Mechanistic Insights to Biomarker Identification
Article Menu
Issue 9 (September) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2018, 19(9), 2776; https://doi.org/10.3390/ijms19092776

The Pathways of the iRFP713 Unfolding Induced by Different Denaturants

1
Laboratory of Structural dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave, St. Petersburg 194064, Russia
2
Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, Polytechnicheskaya str., 29, St. Petersburg 194064, Russia
*
Author to whom correspondence should be addressed.
Received: 23 August 2018 / Revised: 11 September 2018 / Accepted: 13 September 2018 / Published: 15 September 2018
(This article belongs to the Section Molecular Biophysics)
Full-Text   |   PDF [5930 KB, uploaded 15 September 2018]   |  

Abstract

Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome RpBphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH+ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH+ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter A, was found to have a different residual structure in the denaturants used. View Full-Text
Keywords: iRFP713; protein unfolding; protein aggregation; intermediate state; guanidinium cation iRFP713; protein unfolding; protein aggregation; intermediate state; guanidinium cation
Figures

Graphical abstract

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Supplementary material

SciFeed

Share & Cite This Article

MDPI and ACS Style

Stepanenko, O.V.; Stepanenko, O.V.; Kuznetsova, I.M.; Turoverov, K.K. The Pathways of the iRFP713 Unfolding Induced by Different Denaturants. Int. J. Mol. Sci. 2018, 19, 2776.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top