Identification and Characterization of an Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

Samuel, Dharmaraj; Kumar, Thallapuranam Krishnaswamy Suresh; Srimathi, Thiagarajan; Hsieh, Hui-Chu; Yu, Chin

doi:10.1074/jbc.m005147200

Cited by 45 publications

(55 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…These observations are suggestive of a molten-globule-like character of the I1 state; a similar type of molten-globule state was reported previously for human acidic fibroblast growth factor (hFGF-1) (47,48). The I2 state has substantial secondary structure and also shows weak ANS binding, which suggests that this state is a partly condensed state with a small solvent-accessible non-polar cluster.…”

Section: Discussionsupporting

confidence: 82%

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Shukla

Singh

Vispute

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

Cyclophilin catalyzes the ubiquitous process ''peptidyl-prolyl cis-trans isomerization,'' which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1 H-15 N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

show abstract

Section: Discussionsupporting

confidence: 82%

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Shukla

Singh

Vispute

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…The GdnHCl-induced unfolding of hFGF-1 is non-cooperative (38). GdnHCl-induced unfolding of hFGF-1 was monitored by steady-state fluorescence (based on the 350/308 nm fluorescence changes) and far UV CD (using ellipticity changes at 228 nm).…”

Section: Accumulation Of An Equilibrium Unfolding Intermediate State(s)mentioning

confidence: 99%

“…Middaugh and co-workers (36,37) showed that hFGF-1 exists in a partially structured state(s) that exhibits high affinity to bind to negatively charged phospholipid vesicles. Recently, Samuel et al (38) identified and characterized an obligatory partially unfolded intermediate state in the GdnHCl unfolding pathway of hFGF-1. In the present study, we investigate the structure and dynamic features of this obligatory intermediate state of hFGF-1 using a variety of techniques including multidimensional NMR.…”

mentioning

confidence: 99%

Characterization of the Structure and Dynamics of a Near-native Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

Srimathi

Kumar

Chi

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. 15 N spin relaxation experiments show that many residues located in ␤-strands IX, X, and XI exhibit conformational motions in the micro-to millisecond time scale. Analysis of 15 N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the ␤-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.Understanding the mechanism by which a disordered polypeptide chain folds to a unique three-dimensional structure is one of the major challenges in molecular structural biology (1-4). It is elucidated that folding of moderate size proteins (Ͼ10-kDa) proceeds along well defined intermediates similar to a chemical reaction (5-9). Formation of partially folded intermediate states during the protein-folding process is believed to aid in avoiding search of large conformation space on the energy landscape (10, 11). In addition to their role in protein folding, partially structured intermediates are proposed to be involved in a number of biological processes such as interaction with molecular chaperones, translocation across biological membranes, formation of amyloid, and dissociation of supramolecular complexes (12)(13)(14). Thus, investigation of the structural features of equilibrium folding/unfolding intermediates would not only enhance our understanding of the mechanism of protein folding but is also expected to provide strong clues on the interplay of molecular forces in many natural and disease-related processes.Dynamic information about a protein on different time and length scales is important to gain useful knowledge on the mechanism of the protein folding process (15-17). In addition, investigation of the conformational dynamics in the partially structured state(s) can provide valuable information about the interaction potential energy landscape, which is crucial for understanding protein stability and rationalizing protein design (18). In this context, NMR spectroscopy is an apt technique to probe protein folding landscape, because it provides a unique opportunity to study the conformational dynamics of unfolded, partially folded, and native state(s) at the level of individual amino acids (3, 19 -21). Amide proton exchange kinetics and 15 N-relaxation measurements (using NMR) have been successfully used to probe the structural and dynamic features of partially structured intermediate state(s) of proteins (17,(22)(23)(24)(25).Acidic human fibroblast growth factor (hFGF-1) 1 is a 16-kDa, all ␤-sheet protein, devoid of disulfide bonds. The secondary structural elements in the protein include 12 ␤-strands arranged antiparallel into a ␤-barrel architecture (Fig. 1 (26 -31). hFGF-1 is a potent mitogen and plays crucial roles in important cellular processes such as morphogenesis, develo...

show abstract

“…7, inset). The fluorescence of the lone tryptophan residue located at position 121 is significantly quenched in the native state of the protein (43)(44)(45)(46). This quenching effect is attributed to the presence of imidazole and pyrrole groups in the vicinity of the indole ring of the tryptophan (Trp-121) residue (31).…”

Section: Fig 6 Aggregation Induced By Pre-formed Fibrils (Formed Inmentioning

confidence: 99%

Amyloid-like Fibril Formation in an All β-Barrel Protein

Sampath

Kumar

Rajalingam

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a ϳ15-kDa, all ␤-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-␤ structure with an average diameter of 10 -15 Å. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native ␤-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native ␤-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be "sticky" with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular ␤-sheet formation among the extended, linear ␤-strands in the protein.Protein aggregation is a problem of importance not only in biotechnology but also in health-related industries (1, 2). Globular proteins in aqueous solution often tend to aggregate under a variety of conditions of concentration, temperature, pH, and ionic strength (3-6). The morphology of aggregates formed varies considerably and ranges from amorphous forms to highly structured fibrils (7-9). Structured fibrils formed in vitro closely resemble the highly organized amyloid fibrils found in association with a variety of human disorders, including Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, and type II diabetes (10 -19). Several studies show proteins that are apparently unrelated in sequence and, in their native conformation, aggregate into fibrils that have characteristic amyloid-like structural and histological features (16, 20 -26). Recently, Bucciantini et al. (27) demonstrated that amyloid-like fibrils of SH3 domain (from bovine phosphatidylinositol 3-kinase) induced in vitro in 25% (v/v) 2,2,2-trifluoro ethanol (TFE) 1 (under appropriate conditions) are cytotoxic to fibroblast NIH3T3 cells. The amyloid fibrils generated ex vivo are observed to seed fibrillate in cultured NIH3T3 cells (23). Therefore, it is now increasingly believed that amyloid represents a generic form of polypeptide conformation, and all peptides/proteins have the potential to form amyloid-like fibrils under appropriate conditions (21-27).The...

show abstract

Identification and Characterization of an Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

Abstract: The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all ␤-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy.

Cited by 45 publications

References 38 publications

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Characterization of the Structure and Dynamics of a Near-native Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

Amyloid-like Fibril Formation in an All β-Barrel Protein

Contact Info

Product

Resources

About