scholarly journals The Crucial Role of Conserved Intermolecular H-bonds Inaccessible to the Solvent in Formation and Stabilization of the TL5·5 SrRNA Complex

2005 ◽  
Vol 280 (16) ◽  
pp. 16151-16156 ◽  
Author(s):  
George M. Gongadze ◽  
Alexey P. Korepanov ◽  
Elena A. Stolboushkina ◽  
Natalia V. Zelinskaya ◽  
Anna V. Korobeinikova ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Ribosomal Proteins ◽  
Rna Binding ◽  
Thermus Thermophilus ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Intermolecular Hydrogen Bonds ◽  
Conserved Residues ◽  
The Stability

Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins,Escherichia coliL25 andThermus thermophilusTL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5·5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals Structure and Photophysics of 2-(2‘-Pyridyl)benzindoles:  The Role of Intermolecular Hydrogen Bonds

2007 ◽  
Vol 111 (45) ◽  
pp. 11400-11409 ◽  
Author(s):  
Irina Petkova ◽  
Maria S. Mudadu ◽  
Ajay Singh ◽  
Randolph P. Thummel ◽  
Ivo H. M. van Stokkum ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Intermolecular Hydrogen Bonds

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Biochimie ◽  
2005 ◽  
Vol 87 (12) ◽  
pp. 1056-1064 ◽  
Author(s):  
Lilian González-Segura ◽  
Roberto Velasco-García ◽  
Enrique Rudiño-Piñera ◽  
Carlos Mújica-Jiménez ◽  
Rosario A. Muñoz-Clares
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Homology Modeling ◽  
Aldehyde Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
The Stability

scholarly journals Expanding Role of Ubiquitin in Translational Control

2020 ◽  
Vol 21 (3) ◽  
pp. 1151 ◽  
Author(s):  
Shannon E. Dougherty ◽  
Austin O. Maduka ◽  
Toshifumi Inada ◽  
Gustavo M. Silva
Keyword(s):  
Translational Control ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Protein Quality ◽  
Rna Binding Proteins ◽  
Ubiquitin Chain ◽  
Molecular Aspects ◽  
And Function

The eukaryotic proteome has to be precisely regulated at multiple levels of gene expression, from transcription, translation, and degradation of RNA and protein to adjust to several cellular conditions. Particularly at the translational level, regulation is controlled by a variety of RNA binding proteins, translation and associated factors, numerous enzymes, and by post-translational modifications (PTM). Ubiquitination, a prominent PTM discovered as the signal for protein degradation, has newly emerged as a modulator of protein synthesis by controlling several processes in translation. Advances in proteomics and cryo-electron microscopy have identified ubiquitin modifications of several ribosomal proteins and provided numerous insights on how this modification affects ribosome structure and function. The variety of pathways and functions of translation controlled by ubiquitin are determined by the various enzymes involved in ubiquitin conjugation and removal, by the ubiquitin chain type used, by the target sites of ubiquitination, and by the physiologic signals triggering its accumulation. Current research is now elucidating multiple ubiquitin-mediated mechanisms of translational control, including ribosome biogenesis, ribosome degradation, ribosome-associated protein quality control (RQC), and redox control of translation by ubiquitin (RTU). This review discusses the central role of ubiquitin in modulating the dynamism of the cellular proteome and explores the molecular aspects responsible for the expanding puzzle of ubiquitin signals and functions in translation.

Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

2016 ◽  
Vol 42 (2) ◽  
Author(s):  
Hasnain Hussain ◽  
Nikson Fatt Ming Chong
Keyword(s):  
Catalytic Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Qualitative And Quantitative Analysis ◽  
Conserved Residues ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Catalytic Network ◽  
And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

scholarly journals Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

2014 ◽  
Vol 58 (10) ◽  
pp. 6101-6110 ◽  
Author(s):  
Angela Corona ◽  
Francesco Saverio Di Leva ◽  
Sylvain Thierry ◽  
Luca Pescatori ◽  
Giuliana Cuzzucoli Crucitti ◽  
...  
Keyword(s):  
Rational Design ◽  
Acid Ethyl Ester ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Conserved Residues ◽  
Essential Function ◽  
Catalytic Motif ◽  
Hiv 1 ◽  
Micromolar Range

ABSTRACTHIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg2+-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.

scholarly journals Role of Hydrophobic Residues in the Central Ectodomain of gp41 in Maintaining the Association between Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Subunits gp120 and gp41

2004 ◽  
Vol 78 (9) ◽  
pp. 4921-4926 ◽  
Author(s):  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Human Immunodeficiency Virus ◽  
Conformational Changes ◽  
Envelope Glycoprotein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Virus Envelope ◽  
Hydrophobic Residues ◽  
Immunodeficiency Virus ◽  
Mediate Cell ◽  
The Stability

ABSTRACT The interaction between the gp120 and gp41 subunits of the human immunodeficiency virus envelope glycoprotein serves to stabilize the virion form of the complex and to transmit receptor-induced conformational changes in gp120 to trigger the membrane fusion activity of gp41. In this study, we used site-directed mutagenesis to identify amino acid residues in the central ectodomain of gp41 that contribute to the stability of the gp120-gp41 association. We identified alanine mutations at six positions, including four tryptophan residues, which result in mutant envelope glycoprotein complexes that fail to retain gp120 on the cell surface. These envelope glycoproteins readily shed their gp120 and are unable to mediate cell-cell fusion. These findings suggest an important role for the conserved bulky hydrophobic residues in stabilizing the gp120-gp41 complex.

scholarly journals Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

2010 ◽  
Vol 76 (17) ◽  
pp. 6001-6005 ◽  
Author(s):  
Yong-Sheng Tian ◽  
Ai-Sheng Xiong ◽  
Jing Xu ◽  
Wei Zhao ◽  
Feng Gao ◽  
...  
Keyword(s):  
Genomic Library ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Construction Process ◽  
Ochrobactrum Anthropi ◽  
Gene Encoding ◽  
Phosphate Synthase

ABSTRACT Applying the genomic library construction process and colony screening, a novel aro A gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.

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