Identification of 1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic Acid Binding Sequences in α-Crystallin

Sharma, K. Krishna; Kumar, Gyanendra; Murphy, Anne; Kester, Kathryn

doi:10.1074/jbc.273.25.15474

Cited by 125 publications

(114 citation statements)

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“…PsHsp18.1 single site mutants were generated using the Strategene quick change method (Stratagene) using a plasmid containing PsHsp18.1 with a C-terminal Strep tag (36). An amber stop codon was introduced at the desired position for Bpa incorporation and the mutant construct was transformed into BL21 E. coli cells along with the pSup-BpaRS-6TRN plasmid which was a generous gift from Dr. Peter Schultz (Scripps Institute, CA) (24). E. coli cells were grown in 2XYT media containing 1 mM Bpa (Bachem Americas, Inc.) and purified to Ͼ95% homogeneity by conventional methods (27).…”

Section: Methodsmentioning

confidence: 99%

“…However, these data do not distinguish between disruption of substrate interaction sites on the N-terminal arm, versus perturbation of some other sHSP property, such as oligomer integrity, which then indirectly impacts chaperone activity. Other data suggest there are additional substrate binding sites on the ␣-crystallin domain, particularly in certain regions involved in oligomer contacts (10,14,24,25).…”

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confidence: 99%

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Substrate binding site flexibility of the small heat shock protein molecular chaperones

Jaya

García

Vierling

2009

Proc. Natl. Acad. Sci. U.S.A.

217

190

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Small heat shock proteins (sHSPs) serve as a first line of defense against stress-induced cell damage by binding and maintaining denaturing proteins in a folding-competent state. In contrast to the well-defined substrate binding regions of ATP-dependent chaperones, interactions between sHSPs and substrates are poorly understood. Defining substrate-binding sites of sHSPs is key to understanding their cellular functions and to harnessing their aggregation-prevention properties for controlling damage due to stress and disease. We incorporated a photoactivatable cross-linker at 32 positions throughout a well-characterized sHSP, dodecameric PsHsp18.1 from pea, and identified direct interaction sites between sHSPs and substrates. Model substrates firefly luciferase and malate dehydrogenase form strong contacts with multiple residues in the sHSP N-terminal arm, demonstrating the importance of this flexible and evolutionary variable region in substrate binding. Within the conserved ␣-crystallin domain both substrates also bind the ␤-strand (␤7) where mutations in human homologs result in inherited disease. Notably, these binding sites are poorly accessible in the sHSP atomic structure, consistent with major structural rearrangements being required for substrate binding. Detectable differences in the pattern of cross-linking intensity of the two substrates and the fact that substrates make contacts throughout the sHSP indicate that there is not a discrete substrate binding surface. Our results support a model in which the intrinsicallydisordered N-terminal arm can present diverse geometries of interaction sites, which is likely critical for the ability of sHSPs to protect efficiently many different substrates.alpha-crystallin ͉ cross-linking ͉ intrinsic disorder ͉ P-benzoylphenylalanine ͉ protein-protein interactions

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Jaya

García

Vierling

2009

Proc. Natl. Acad. Sci. U.S.A.

217

190

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“…The C-terminal extension of αB-crystallin may be responsible for this flexibility. Recently, Tanaka et al [139] have examined the fibril-forming propensity and chaperone activity of peptides from αA-crystallin, specifically those corresponding to the putative chaperone-binding regions of the protein previously identified by Sharma et al [136,137]. Tanaka et al [139] found that one of these peptide (70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88) inhibited Aβ fibril formation but promoted the aggregation of insulin.…”

Section: In Vitro and In Vivo Formation Of Amyloid Fibrils By Crystalmentioning

confidence: 99%

“…Hydrophobic sites in α-crystallin are thought to be responsible for its chaperone activity [45,[134][135][136][137] and hydrophobic peptides isolated from αA-crystallin (residues 70-88) [137] and αB-crystallin (residues 73-92 and 131-141) [24, 138] possess chaperone activity against amorphously aggregating target proteins. The chaperone activity of these peptides results from their ability to bind independently to target proteins [137].…”

Section: The Region(s) Of α-Crystallin Responsible For Target Proteinmentioning

confidence: 99%

Crystallin proteins and amyloid fibrils

Ecroyd

Carver

2008

Cell. Mol. Life Sci.

220

234

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Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, alpha-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer's and Parkinson's). In this review, the literature on the interaction of alpha B-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials. Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, α-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer's and Parkinson's). In this review, the literature on the interaction of αB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials.

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“…Although no common sequence has been identified among chaperones of different family of proteins, some common features emerges. Chaperones have distinct hydrophilic and hydrophobic domains to enhance solubility and to bind lipophilic molecules (13)(14)(15). Many of them have a characteristic micelle-like-associated structure.…”

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confidence: 99%

Molecular Chaperone-like Properties of an Unfolded Protein, αs-Casein

Bhattacharyya¹,

Das

1999

Journal of Biological Chemistry

177

159

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All molecular chaperones known to date are well organized, folded protein molecules whose three-dimensional structure are believed to play a key role in the mechanism of substrate recognition and subsequent assistance to folding. A common feature of all protein and nonprotein molecular chaperones is the propensity to form aggregates very similar to the micellar aggregates. In this paper we show that ␣ s -casein, abundant in mammalian milk, which has no well defined secondary and tertiary structure but exits in nature as a micellar aggregate, can prevent a variety of unrelated proteins/ enzymes against thermal-, chemical-, or light-induced aggregation. It also prevents aggregation of its natural substrates, the whey proteins. ␣ s -Casein interacts with partially unfolded proteins through its solvent-exposed hydrophobic surfaces. The absence of disulfide bridge or free thiol groups in its sequence plays important role in preventing thermal aggregation of whey proteins caused by thiol-disulfide interchange reactions. Our results indicate that ␣ s -casein not only prevents the formation of huge insoluble aggregates but it can also inhibit accumulation of soluble aggregates of appreciable size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. This protein seems to have some characteristics of cold shock protein, and its chaperone-like activity increases with decrease of temperature.

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Identification of 1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic Acid Binding Sequences in α-Crystallin

Abstract: The hydrophobic binding sites in ␣-crystallin were evaluated using fluorescent probes 1,1-bi(4-anilino)naphthalenesulfonic acid (

Cited by 125 publications

References 41 publications

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Crystallin proteins and amyloid fibrils

Molecular Chaperone-like Properties of an Unfolded Protein, αs-Casein

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