A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

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Differential binding affinity of tau repeat region R2 with neuronal-specific β-tubulin isotypes

Scientific Reports ◽

10.1038/s41598-019-47249-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Vishwambhar Vishnu Bhandare ◽

Bajarang Vasant Kumbhar ◽

Ambarish Kunwar

Keyword(s):

Binding Affinity ◽

Repeat Region ◽

Tubulin Isotypes ◽

Differential Binding ◽

Β Tubulin

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Binding of Radiolabeled Folate and 5-Methyltetrahydrofolate to Cow's Milk Folate Binding Protein at pH 7.4 and 5.0. Relationship to Concentration and Polymerization Equilibrium of the Purified Protein

Bioscience Reports ◽

10.1023/a:1015576522416 ◽

2001 ◽

Vol 21 (6) ◽

pp. 733-743 ◽

Cited By ~ 9

Author(s):

Jan Holm ◽

Steen Ingemann Hansen

Keyword(s):

Ligand Binding ◽

Binding Affinity ◽

Conformational Changes ◽

Protein Conformation ◽

Binding Protein ◽

Cow’S Milk ◽

Folate Binding Protein ◽

Cow's Milk ◽

Polymerization Equilibrium ◽

Methyl Tetrahydrofolate

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 μM) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.

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Identification of a rabbit liver cytosolic binding protein for human growth hormone

Biochemical Journal ◽

10.1042/bj2210617 ◽

1984 ◽

Vol 221 (3) ◽

pp. 617-622 ◽

Cited By ~ 24

Author(s):

S I Ymer ◽

J L Stevenson ◽

A C Herington

Keyword(s):

Growth Hormone ◽

Binding Affinity ◽

Binding Protein ◽

Physiological Role ◽

Rabbit Liver ◽

Precipitation Method ◽

Affinity Column ◽

Separation Procedure ◽

Binding Characteristics ◽

Membrane Bound

A specific growth hormone (GH) binding protein of Mr approx. 100000 has been demonstrated in the cytosolic fraction (200000g supernatant) of pregnant-rabbit liver by gel filtration techniques. This binding species was detectable by a standard charcoal separation procedure but not by the widely used poly(ethylene glycol) precipitation method. The GH binding protein had similar binding characteristics to those of classical membrane-bound GH receptors. The kinetics of association and dissociation, binding affinity (2.56×10(9)1/mol) and hormonal specificity have been established. There appears to be equal or greater amounts of GH binding protein in the cytosol than in the membrane fraction. The presence of the GH binding protein in rabbit liver cytosol was substantiated by its selective purification on a GH-Affigel 15 affinity column. This technique has resulted in a 200-300-fold purification with no substantial change in binding affinity. The ability of a concanavalin A-Sepharose affinity column to also bind the cytosolic binding protein indicates that, like the membrane-bound GH receptor, it is a glycoprotein. This is the first report of a cytosolic binding protein for GH and raises important questions regarding its potential physiological role in the mechanism of action of GH.

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