Protein–Protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study

Mishra, Kamini; Chakrabarti, Abhijit; Das, Puspendu K.

doi:10.1021/acs.jpcb.7b04503

Cited by 12 publications

(17 citation statements)

References 29 publications

(39 reference statements)

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“…Basic sequence alignment and crystal structure comparison reveals that the spectrin repeat domains share a similar fold and moderate homology to the α‐globin chaperone AHSP (Feng et al, ). We have hypothesized that α‐globin may act as a major client for the chaperone activity of spectrin based on both its selective binding and high affinity (Basu & Chakrabarti, ; Mishra, Chakrabarti, & Das, ). This selective chaperone activity may have significance in hemoglobin disease states, notably in β‐thalasseimas where it is seen that free α‐globin causes cellular toxicity and disease manifestations (Cao & Galanello, ).…”

Section: Discussionmentioning

confidence: 99%

“…We have hypothesized that α-globin may act as a major client for the chaperone activity of spectrin based on both its selective binding and high affinity (Basu & Chakrabarti, 2015;Mishra, Chakrabarti, & Das, 2017). This selective chaperone activity may have significance in hemoglobin disease states, notably in β-thalasseimas where it is seen that free α-globin causes cellular toxicity and disease manifestations (Cao & Galanello, 2010).…”

Section: Discussionmentioning

confidence: 99%

Section: Isolation Of Human Erythroid Spectrinmentioning

confidence: 99%

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Localizing the chaperone activity of erythroid spectrin

Bose

Chakrabarti

2019

Cytoskeleton

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Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone‐like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self‐association domain. Here we probe the location and molecular origin of the chaperone activity of multi‐domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α‐ and β‐globin as well as enzyme refolding assays using alkaline phosphatase and α‐glucosidase show that the chaperone activity is not only localized in the self‐association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi‐repeat proteins, possibly implicating that the large family of “spectrin‐repeat” domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α‐ over β‐globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme‐refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1‐anilinonaphthalene‐8‐sulfonic acid) and Prodan (6‐propionyl‐2[dimethylamino]‐naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self‐association domain.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Isolation Of Human Erythroid Spectrinmentioning

confidence: 99%

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Localizing the chaperone activity of erythroid spectrin

Bose

Chakrabarti

2019

Cytoskeleton

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“…Basic sequence alignment and crystal structure comparison reveals that the spectrin repeat domains share a similar fold and moderate homology to the α-globin chaperone AHSP (71). We have hypothesised that α-globin may act as a major client for the chaperone activity of spectrin based on both its selective binding and high affinity (21,72).…”

Section: Discussionmentioning

confidence: 99%

“…Human blood samples were collected from healthy volunteers with proper informed consent. Blood samples were obtained from Ramkrishna Mission Seva Pratisthan Hospital, Kolkata, India, with informed written consent of the patients following the guidelines of the Institutional Ethical Committee as elaborated earlier (72,75,76) Dimeric human erythroid spectrin was isolated from clean white RBC ghost membranes following protocol elaborated in earlier studies (77). Briefly, RBCs were pelleted by centrifugation, washed with PBS containing 5 mM sodium phosphate, 155 mM NaCl, 1mM EDTA, pH 8.0 and lysed in hypotonic lysis buffer containing 5mM sodium phosphate, 1mM EDTA and 20 µg/ml PMSF.…”

Section: Isolation Of Human Erythroid Spectrinmentioning

confidence: 99%

Localizing the chaperone activity of erythroid spectrin

Bose

Chakrabarti

2019

Preprint

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Spectrin, the major protein of the RBC membrane skeleton has canonically been thought to only serve a structural function. We have described a novel chaperone-like property of spectrin and have shown that it is able to prevent the aggregation of other proteins such as alcohol dehydrogenase, insulin and free globin chains. We have tried to localize the molecular origin of chaperonelike activity in multi-domain spectrin by using recombinant spectrin fragments and investigating individual domains. We have characterized the recombinant domains using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Hydrophobic ligands Prodan (6propionyl-2[dimethylamino]-naphthalene) and ANS (1-anilinonaphthalene-8-sulfonic acid) binding has been used to probe the hydrophobicity of the recombinant domains and it is seen that all domains have surface exposed hydrophobic patches; and in accordance with our previous hypothesis only the reconstituted self-association domain binds Prodan. Recombinant domains display comparable chaperone potential in preventing protein aggregation; and substrate selectivity of α-over β-globin is seen. Enzyme refolding studies show alternate pathways of chaperone action. Our current study points to the presence of hydrophobic patches on the surface of these domains as the source of the chaperone activity of spectrin, as notably seen in the self-association domain. There is no one domain largely responsible for the chaperone activity of spectrin; rather all domains appear to contribute equally, such that the chaperone activity of spectrin seems to be a linear sum of the individual activities of the domains.

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Construction of Protein Probe with a His‐tag and an Electron‐transfer Peptide for a Target Protein Sensing

et al. 2020

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A protein probe with an electron‐transfer peptide and a His‐tag was designed to electrochemically sense a target protein. We selected tyrosine‐rich (Y4C) and tryptophan‐rich (W4C) peptides for use as electron‐transfer agents. The peak for oxidation was based on the oxidations of the phenolic hydroxy groups in Y4C and on the indole rings in W4C. Asialofetuin (ASF) with galactose residues was the protein probe, and a galactose recognition protein, soybean agglutinin (SBA), was the target protein. A protein probe composed of an amino acid and carbohydrate residue was expected to be biocompatible. When voltammetric measurements were performed using a glassy carbon electrode, the oxidation peaks of H6Y4C and ASF‐H6Y4C appeared at the same potential. The peak current of ASF‐H6Y4C was 4‐fold that of H6Y4C because of the stronger adsorption of ASF‐H6Y4C onto the electrode. The electrode response of ASF‐H6Y4C with SBA was half that of ASF‐H6Y4C alone. By contrast, the peak current of ASF‐Y4CH6 was higher than that of ASF‐H6Y4C, which was the result of a greater degree of contact between the Y4C moieties and an electrode. On the other hand, the voltammetric behaviors of ASF with W4C and a His‐tag were similar to those with Y4C and a His‐tag. The sensitivity of SBA using ASF‐Y4CH6 was at the 10−13 M level. To confirm the function of the sensing system, measurements were performed in human serum with SBA and ASF‐Y4CH6. When SBA was added, the serum had a concentration that ranged between 5.0×10−13 and 4.0×10−12 M, and the amount of SBA that could be recovered ranged from 97 to 101%. Consequently, this system could be applied to the detection of SBA in serum.

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Protein–Protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study

Cited by 12 publications

References 29 publications

Localizing the chaperone activity of erythroid spectrin

Localizing the chaperone activity of erythroid spectrin

Localizing the chaperone activity of erythroid spectrin

Construction of Protein Probe with a His‐tag and an Electron‐transfer Peptide for a Target Protein Sensing

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