Interaction of guanine ligands with ribonuclease T1

Walz, Frederick G.; Hooverman, Lois L.

doi:10.1021/bi00748a006

Cited by 35 publications

(44 citation statements)

References 20 publications

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“…The dissociation constant for 3'-GMP and barnase was measured using UV difference spectroscopy. The UV difference spectrum of the barnase-3'-GMP complex relative to free barnase and 3'-GMP has a maximum at 290 nm and a minimum at 243 nm, similar to that observed previously for RNase T1 (Walz & Hooverman, 1973). The difference in absorbance at 290 and 243 nm ( = 290 -AAj4z) was measured upon titrating a solution of barnase (20>iM protein, 59.8 mM acetic acid/40.2 mM sodium acetate, pH 4.5) with a concentrated solution of 3'-GMP [disodium salt (Sigma) in H2O].…”

Section: Methodssupporting

confidence: 86%

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Meiering¹,

Bycroft²,

Lubienski³

et al. 1993

Biochemistry

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The binding of 3'-GMP to the ribonuclease, barnase, has been studied using heteronuclear 2D and 3D NMR spectroscopy. The 1H and 15N NMR spectra of barnase complexed with 3'-GMP have been assigned. 2D and 3D NOESY spectra have been used to identify inter- and intramolecular NOEs, and a solution structure for the barnase-3'-GMP complex has been calculated. The position of the guanine ring of the ligand is reasonably well defined in the structures. The guanine ring forms hydrogen bonds with the NH protons of Ser57 and Arg59. These residues are located in a loop that is conserved among the microbial guanine-specific ribonucleases. The 2'-hydroxyl of 3'-GMP is close to His102 and Glu73, which have been shown to be involved in catalysis. The phosphate group of 3'-GMP is close to a number of positively charged residues that have also been shown to be important for activity. The position of the sugar moiety of 3'-GMP is less well defined in the structures. Structures calculated for the complex could not simultaneously satisfy all the observed intermolecular NOEs for the sugar protons, suggesting that the sugar samples several conformations when bound to barnase. The binding of 3'-GMP to barnase in solution is similar to that observed in the crystal structures of nucleotides bound to related ribonucleases. 3'-GMP binding causes no major conformational change in barnase. In contrast to the small structural changes that occur, there is a significant decrease in the rates of hydrogen/deuterium exchange and aromatic ring rotation in the active site of barnase upon ligand binding.

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Section: Methodssupporting

confidence: 86%

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Meiering¹,

Bycroft²,

Lubienski³

et al. 1993

Biochemistry

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“…The RNase Ti was a gift of Professor F. G. Walz of Kent State University, prepared by the method of Hooverman (27). Gel electrophoresis exhibited a single band, and the UV absorption spectrum showed that there was no observable contamination.…”

Section: Methodsmentioning

confidence: 99%

Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study

Chen¹,

Longworth²,

Fleming³

1987

Biophysical Journal

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The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A.

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“…GpcU binding with RNase T 1 . Ribonuclease T 1 (Gln25 allozyme) was purified as previously described and its concentration was determined with a molar absorptivity at 278 nm ( 278 nm ) of 2.1 × 10 4 M -1 cm -1 (24). The concentration of GpcU was determined with the molar absorptivity for GpU [ 280 nm ) 1.06 × 10 4 M -1 cm -1 (25)].…”

Section: Preparation Of Gpcumentioning

confidence: 99%

“…The concentration of GpcU was determined with the molar absorptivity for GpU [ 280 nm ) 1.06 × 10 4 M -1 cm -1 (25)]. Binding was studied at pH 5.5 using ultraviolet difference spectroscopy (24). The maximum difference absorbance occurred at 291 nm and values of the difference absorbance, ∆A, at this wavelength were determined for a fixed total concentration of the enzyme ([E] 0 ) 3.43 × 10 -3 M) in the presence of variable total concentrations of GpcU ([L] 0 ; (0.479-2.21) × 10 -4 M).…”

Section: Preparation Of Gpcumentioning

confidence: 99%

Three-Dimensional Structure of Ribonuclease T₁ Complexed with an Isosteric Phosphonate Substrate Analogue of GpU: Alternate Substrate Binding Modes and Catalysis^,

et al. 1999

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The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

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Interaction of guanine ligands with ribonuclease T1

Cited by 35 publications

References 20 publications

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study

Three-Dimensional Structure of Ribonuclease T₁ Complexed with an Isosteric Phosphonate Substrate Analogue of GpU: Alternate Substrate Binding Modes and Catalysis^,

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Interaction of guanine ligands with ribonuclease T1

Cited by 35 publications

References 20 publications

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study

Three-Dimensional Structure of Ribonuclease T1 Complexed with an Isosteric Phosphonate Substrate Analogue of GpU: Alternate Substrate Binding Modes and Catalysis,

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Three-Dimensional Structure of Ribonuclease T₁ Complexed with an Isosteric Phosphonate Substrate Analogue of GpU: Alternate Substrate Binding Modes and Catalysis^,