A Pulsed-Field Gradient NMR Study of Bovine Pancreatic Trypsin Inhibitor Self-Association

Ilyina, Elena; Roongta, Vikram; Pan, Hong; Woodward, Clare; Mayo, Kevin H.

doi:10.1021/bi9622229

Cited by 63 publications

(66 citation statements)

References 18 publications

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“…For example, when the concentration of PDE␥ was lowered from 350 to 90 M, the Ds of PDE␥ increased from 0.92 ϫ 10 Ϫ6 cm 2 ⅐s Ϫ1 to 1.19 ϫ 10 Ϫ6 cm 2 ⅐s Ϫ1 . The measured Ds did not change when the concentration of PDE␥ was lowered from 90 to 45 M. These results suggest that PDE␥ exists as an oligomer at higher concentrations but is monomeric at Ͻ90 M. Furthermore, comparison of the diffusion coefficient for monomeric PDE␥ (10 kDa, Ds ϭ 1.2 ϫ 10 Ϫ6 cm 2 ⅐s Ϫ1 ) with that for monomeric lysozyme (14 kDa, Ds ϭ 1.11 ϫ 10 Ϫ6 cm 2 ⅐s Ϫ1 ) (21), suggested that the structure of PDE␥ in solution is relatively compact, despite its dynamic state.…”

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confidence: 80%

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Song

Guo

Muradov

et al. 2008

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

123

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The retinal phosphodiesterase (PDE6) inhibitory ␥-subunit (PDE␥) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic ␣␤-subunits of PDE6 and the ␣-subunit of transducin (␣t). Detailed structural analysis of PDE␥ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDE␥, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDE␥46-87 when bound to ␣t in the transitionstate complex. NMR mapping of the interaction between PDE␥46-87 and the chimeric PDE5/6 catalytic domain confirmed that Cterminal residues 74 -87 of PDE␥ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to ␣t, is left free at this stage. These results indicate that the interaction between PDE␥ and ␣t during the phototransduction cascade involves the selection of preconfigured transient conformations.NMR spectroscopy ͉ protein recognition ͉ transient structure ͉ visual transduction P hototransduction, the primary event of vision is critically regulated by the ␥-subunit of cGMP phosphodiesterase (PDE␥). In the dark, by binding tightly to the catalytic ␣␤-subunits of cGMP PDE (PDE6) PDE␥ keeps PDE6 inactive; yet upon photoexcitation, interaction of PDE␥ with the ␣-subunit of activated transducin (␣ t ) relieves its inhibitory constraint on PDE6. For termination of phototransduction, PDE␥ interacts with both ␣ t and RGS9, a regulator of G protein signaling, to accelerate ␣ t GTPase activity (1). Beyond phototransduction, the importance of PDE␥ in photoreceptor cell viability is demonstrated by rapid retinal degeneration in the PDE␥-knockout mouse (2), and growing evidence suggests that PDE␥ also interacts with some signaling proteins in nonphototransduction pathways (3).Biochemical studies have shown that the central polycationic region (residues 24-45) and the C-terminal region of PDE␥ constitute two distinct sites of interactions with ␣ t or PDE6 (4). The crystal structure of a ternary complex representing a partial model for the GTPase-activating protein (GAP) complex, consisting of a fragment of PDE␥ (residues 46-87), ␣ t chimera (␣ t/i1 ), and the catalytic core of RGS9, revealed that the C-terminal region of PDE␥ contains three discontinuous helices (5). PDE␥ 46-87 interacts with ␣ t in a GTP-dependent manner, mainly through residues in these helices or their linkers, and the indole side chain of W70 plays the key role in this interaction. Residue V66 of PDE␥ interacts with RGS9, which further tightens the association between ␣ t and RGS9. The C-terminal region of PDE␥ also interacts with the catalytic domain of PDE6; however, studies have suggested that this interaction involves the 11 C-terminal residues of PDE␥, a region distinct from the ␣ t interaction site (6).It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of...

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mentioning

confidence: 80%

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Song

Guo

Muradov

et al. 2008

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

123

View full text Add to dashboard Cite

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“…The linear plots with similar slopes reflect that the oligomerization states of the monomer and the oligomer do not change significantly over the temperature range studied. 30 The ratio of the diffusion coefficients of the oligomer and the monomer (D oligomer /D monomer ) helps establish the oligomerization state. 28a,32 If both the monomer, and the oligomer are envisioned as compact (spherical) structures, then D oligomer /D monomer should be 0.63 for a tetramer and 0.79 for a dimer.…”

Section: Design Synthesis and Study Of Macrocyclic Peptide 3amentioning

confidence: 99%

Macrocyclic β-Sheet Peptides That Mimic Protein Quaternary Structure through Intermolecular β-Sheet Interactions

2007

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This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through β-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide β-strand, two Hao β-strand mimics [JACS 2000, 122, 7654] joined by one additional α-amino acid, and two δ-linked ornithine β-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1 H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide selfassociates through edge-to-edge β-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of β-sheet dimers. In this tetrameric β-sheet sandwich, the macrocyclic peptide 3a is folded to form a β-sheet, the β-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R 3 and R 5 of the heptapeptide sequence are important in oligomerization.

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“…Translational diffusion measurements were performed on a Varian Inova 500 NMR spectrometer by using the pulse field gradient (PFG) method as described previously (29). Similar PFG-NMR studies have been done to assess the self-association and complexation of other protein systems (e.g.…”

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confidence: 99%

Heparin Dodecasaccharide Binding to Platelet Factor-4 and Growth-related Protein-α

Михайлов¹,

Young²,

Linhardt³

et al. 1999

Journal of Biological Chemistry

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␣-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-␣ (Gro-␣) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-␣ give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, 15 N/ 1 H heteronuclear single quantum coherence NMR data demonstrate loss of 1 H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-␣ in particular, 1 H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.

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A Pulsed-Field Gradient NMR Study of Bovine Pancreatic Trypsin Inhibitor Self-Association

Cited by 63 publications

References 18 publications

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Macrocyclic β-Sheet Peptides That Mimic Protein Quaternary Structure through Intermolecular β-Sheet Interactions

Heparin Dodecasaccharide Binding to Platelet Factor-4 and Growth-related Protein-α

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