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1.
J Mol Biol ; 252(1): 133-43, 1995 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-7666425

RESUMO

Electrostatic interactions between charged residues and the helix dipole in a protein were investigated by protein engineering methods. In ribonuclease T1, two surface-exposed acidic residues (Glu28 and Asp29) are located near the carboxyl terminus of the alpha-helix between residues 13 and 29. They were replaced, individually and in concert, by the uncharged amides Gln28 and Asn29, and the stabilities of the wild-type protein and its variants were determined as a function of pH. The effects of the two mutations are additive. Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that the deprotonations of Glu28 and Asp29 reduce the free energy of stabilization of folded ribonuclease T1 by about 4 kJ/mol each. This destabilization is probably caused by unfavorable electrostatic interactions of Glu28 and Asp29 with the negative end of the helix dipole. The activation energies for the unfolding of the different variants of ribonuclease T1 change in parallel with the differences in the thermodynamic stability when the pH is varied. This indicates that the unfavorable electrostatic interactions of Glu28 and Asp29 are lost very early in unfolding, and are not present in the activated state of unfolding.


Assuntos
Ribonuclease T1/química , Asparagina/química , Ácido Aspártico/química , Sequência de Bases , Fenômenos Químicos , Físico-Química , Primers do DNA/química , Glutamatos/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Íons , Modelos Moleculares , Dados de Sequência Molecular , Desnaturação Proteica , Proteínas Recombinantes , Ribonuclease T1/ultraestrutura , Termodinâmica
2.
Eur J Biochem ; 211(1-2): 311-6, 1993 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-8425541

RESUMO

The ternary complex formed between RNase T1, guanosine 3',5'-bisphosphate (3',5'-pGp) and Pi crystallizes in the cubic space group I23 with a = 8.706(1) nm. In a previous publication [Lenz, A., Heinemann, U., Maslowska, M. & Saenger, W. (1991) Acta Crystallogr. B47, 521-527], the structure of the complex (in which Pi was not located) was described at a resolution of 0.32 nm. This is now extended to 0.19 nm with newly grown, larger crystals. Refinement with restrained least-squares converged at R = 17.8% for 8027 reflections with [Fo[ > or = 1 sigma ([Fo[); the final model comprises 120 water molecules. 3',5'-pGp is bound to RNase T1 in the anti form, with guanine in the specific recognition site; the 3'-phosphate protrudes into the solvent, and the 5'-phosphate hydrogen bonds with Lys41 O and Asn43 N4. A tetrahedral anion assigned as Pi occupies the catalytic site and hydrogen bonds to the side chains of Tyr38, Glu58, Arg77 and His92. The overall polypeptide fold of RNase T1 in the cubic space group does not differ significantly from that in the orthorhombic space group P2(1)2(1)2(1) except for changes < or = 0.2 nm in loop regions 69-72 and 95-98.


Assuntos
Ribonuclease T1/ultraestrutura , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/ultraestrutura , Ribonuclease T1/metabolismo , Temperatura
3.
J Mol Biol ; 223(4): 1013-28, 1992 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-1311385

RESUMO

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.


Assuntos
Ribonuclease T1/ultraestrutura , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Gráficos por Computador , Cristalografia , Análise Mutacional de DNA , Guanosina Monofosfato/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , Ribonuclease T1/genética , Ribonuclease T1/metabolismo , Relação Estrutura-Atividade , Fagos T/enzimologia , Triptofano/química , Tirosina/química , Água/química , Difração de Raios X
4.
J Mol Biol ; 222(2): 335-52, 1991 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-1960730

RESUMO

The free form of ribonuclease T1 (RNase T1) has been crystallized at neutral pH, and the three-dimensional structure of the enzyme has been determined at 1.5 A nominal resolution. Restrained least-squares refinement yielded an R value of 14.3% for 12,623 structure amplitudes. The high resolution of the structure analysis permits a detailed description of the solvent structure around RNase T1, the reliable rotational setting of several side-chain amide and imidazole groups and the identification of seven disordered residues. Among these, the disordered and completely internal Val78 residue is noteworthy. In the RNase T1 crystal structures determined thus far it is always disordered in the absence of bound guanosine, but not in its presence. A systematic analysis of hydrogen bonding reveals the presence in RNase T1 of 40 three-center and an additional seven four-center hydrogen bonds. Three-center hydrogen bonds occur predominantly in the alpha-helix, where their minor components close 3(10)-type turns, and in beta-sheets, where their minor components connect the peptide nitrogen and carbonyl functions of the same residue. The structure of the free form is compared with complexes of RNase T1 with filled base recognition site and/or catalytic site. Several structural rearrangements occurring upon inhibitor or substrate binding are clearly apparent. In conjunction with the available biochemical knowledge, they are used to describe probable steps occurring early during RNase T1-catalyzed phosphate transesterification.


Assuntos
Ribonuclease T1/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Catálise , Cristalografia , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Água
5.
FEBS Lett ; 290(1-2): 216-20, 1991 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-1655533

RESUMO

Complex of a mutant ribonuclease T1 (Y45W) with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined by X-ray diffraction at 1.7 A resolution. The 2'AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2'GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3'-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2'AMP to the guanine-recognition site is weaker than that to the new binding site.


Assuntos
Monofosfato de Adenosina/metabolismo , Ribonuclease T1/metabolismo , Sequência de Aminoácidos , Aspergillus oryzae/enzimologia , Sítios de Ligação , Cristalografia , Guanosina Monofosfato/metabolismo , Ligação de Hidrogênio , Isomerismo , Modelos Moleculares , Estrutura Molecular , Mutação , Proteínas Recombinantes/ultraestrutura , Ribonuclease T1/ultraestrutura , Relação Estrutura-Atividade , Difração de Raios X
6.
Biochemistry ; 29(27): 6475-80, 1990 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-2119802

RESUMO

The refolding of ribonuclease T1 is dominated by two major slow kinetic phases that show properties of proline isomerization reactions. We report here that the molecular origin of one of these processes is the trans----cis isomerization of the Ser54-Pro55 peptide bond, which is cis in the native protein but predominantly trans in unfolded ribonuclease T1. This is shown by a comparison of the wild type and a designed mutant protein where Ser54 and Pro55 were replaced by Gly54 and Asn55, respectively. This mutation leaves the thermal stability of the protein almost unchanged; however, in the absence of Pro55 one of the two slow phases in folding is abolished and the kinetic mechanism of refolding is dramatically simplified.


Assuntos
Ribonuclease T1/ultraestrutura , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Cinética , Modelos Moleculares , Prolina , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes , Ribonuclease T1/genética , Estereoisomerismo
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