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1.
Acta Crystallogr D Struct Biol ; 80(Pt 7): 464-473, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38860981

ABSTRACT

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.


Subject(s)
Archaeal Proteins , Sulfolobus solfataricus , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Crystallography, X-Ray , Models, Molecular , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/chemistry , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Protein Conformation , Binding Sites , RNA, Transfer, Met/chemistry , RNA, Transfer, Met/metabolism
2.
Mol Biol (Mosk) ; 52(1): 106-111, 2018.
Article in Russian | MEDLINE | ID: mdl-29512642

ABSTRACT

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.


Subject(s)
Bacterial Proteins/chemistry , RNA, Ribosomal, 23S/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Thermus thermophilus/chemistry , Kinetics , Nucleic Acid Conformation , Protein Binding
3.
Biochemistry (Mosc) ; 81(10): 1205-1212, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27908245

ABSTRACT

The crystal structure of the γ-subunit of translation initiation factor 2 from the archaeon Sulfolobus solfataricus (SsoIF2γ) has been solved based on perfectly hemihedral twinned data. The protein was cocrystallized with the 10-fold molar excess of GTP analog (GDPCP) over protein. However, no nucleotide was found in the structure, and the model demonstrated the apo form of the protein. Two slightly different molecules in the asymmetric unit of the crystal are related by the non-crystallographic 2-fold axis and form a tightly associated dimer. This dimer is stabilized by an intermolecular hydrophobic core and hydrogen bonds. Lack of GDPCP in the nucleotide-binding pocket of the γ-subunit and significant excess of dimers over monomers in the crystallization solution suggest that these dimers are the building blocks of the crystal. Contrary to SsoIF2γ monomers, these dimers are able to crystallize in two oppositely oriented slightly different crystal domains, thus forming a twinned crystal. Comparison of crystallization conditions for the twinned and untwinned crystals of apo SsoIF2γ showed that stabilization of the dimers in the solution may be caused by higher sodium salt concentration. Since amino acid residues involved in intermolecular contacts in the dimer are responsible for binding of the γ- and α-subunits within SsoIF2, increase in sodium salt concentration may prevent functioning of SsoIF2 in the cell.


Subject(s)
Peptide Initiation Factors/chemistry , Protein Subunits/chemistry , Sulfolobus solfataricus/chemistry , Crystallography, X-Ray
4.
Biochemistry (Mosc) ; 79(8): 826-35, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25365493

ABSTRACT

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Mutation , RNA, Ribosomal, 5S/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Base Sequence , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Binding , Protein Conformation , RNA, Ribosomal, 5S/chemistry , RNA, Ribosomal, 5S/genetics , Ribosomal Proteins/chemistry , Ribosomes/chemistry
5.
Article in Russian | MEDLINE | ID: mdl-19672222

ABSTRACT

One thousand and thirty patients with proved L(5)/S(1)disc herniations were examined. The dynamics of disease in acute stage was studied using ultrasonography and electromyography in 3 groups of patients: 280 with lumboischialgia, 520 with the root syndrome and 230 controls without back pain. It has been shown that edematous epiduritis and venous stas in the superincumbent spinal movement segment is a main pathogenetic mechanism of acute phase of the disc-radicular conflict. Focal myelopathy is a pathogenetic mechanism of radiculopathy in the disc-recticular conflict. Compression and reflex syndromes are caused by the common mechanisms and their differentiation by clinical presentations has a conditional character.


Subject(s)
Intervertebral Disc Displacement/complications , Low Back Pain/etiology , Lumbar Vertebrae , Nerve Compression Syndromes/etiology , Radiculopathy/complications , Sacrum , Adolescent , Adult , Aged , Diagnosis, Differential , Electromyography , Follow-Up Studies , Humans , Intervertebral Disc Displacement/diagnosis , Low Back Pain/diagnosis , Middle Aged , Nerve Compression Syndromes/diagnosis , Radiculopathy/diagnosis , Severity of Illness Index , Spinal Nerves/diagnostic imaging , Spinal Nerves/pathology , Syndrome , Ultrasonography , Young Adult
6.
Acta Crystallogr D Biol Crystallogr ; 64(Pt 3): 248-56, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18323619

ABSTRACT

The crystal structures of Erwinia carotovora L-asparaginase complexed with L-aspartate and L-glutamate were determined at 1.9 and 2.2 A, respectively, using the molecular-replacement method and were refined to R factors of about 21% in both cases. The positions of the ligands in the active site were located. A comparison of the new structures with the known structures of Escherichia coli L-asparaginase and Er. chrysanthemi L-asparaginase was performed. It was found that the arrangement of the ligands practically coincides in all three enzymes. The peculiarities of the quaternary structure of the enzyme, the possible role of water molecules in the enzyme action and the conformational changes during the catalyzed reaction are discussed.


Subject(s)
Asparaginase/chemistry , Aspartic Acid/chemistry , Glutamic Acid/chemistry , Pectobacterium carotovorum/enzymology , Crystallography, X-Ray , Models, Molecular , Protein Conformation
7.
Mol Biol (Mosk) ; 41(4): 688-96, 2007.
Article in Russian | MEDLINE | ID: mdl-17936990

ABSTRACT

Nine mutant forms of ribosomal proteins L1 from the bacterium Thermus thermophilus and the archaeon Methanococcus jannaschii were obtained. Their crystal structures were determined and analyzed. Earlier determined structure of S179C TthL1 was also thoroughly analyzed. Five from ten mutant proteins reveal essential changes of spatial structure caused by surface point mutation. It proves that for correct studies of biological processes by site-directed mutagenesis it is necessary to determine or at least to model spatial structures of mutant proteins. Detailed comparison of mutant L1 structures with that of corresponding wild type proteins reveals that side chain of a mutated amino acid residue tries to locate like the side chain of the original residue in the wild type protein. This observation helps to model the mutant structures.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Amino Acid Sequence , Crystallography, X-Ray , Methanococcus/metabolism , Molecular Sequence Data , Mutation , Protein Conformation , Thermus thermophilus/metabolism
8.
Mol Biol (Mosk) ; 40(4): 650-7, 2006.
Article in Russian | MEDLINE | ID: mdl-16913224

ABSTRACT

Crystal structures of unbound protein L1 and of its complexes with ribosomal an messenger RNAs are analyzed. It is shown that the values of the apparent association rate constant for L1-RNA depend on conformation of unbound protein L1. It is suggested that L1 binds to rRNA with higher affinity than to mRNA because of additional interactions between domain II of L1 and the loop rRNA region, which is absent in mRNA.


Subject(s)
RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism , Archaeal Proteins/metabolism , Bacterial Proteins/metabolism , Models, Molecular , Protein Binding , Protein Biosynthesis , Protein Conformation , RNA, Archaeal/metabolism , RNA, Bacterial/metabolism
9.
Acta Crystallogr D Biol Crystallogr ; 61(Pt 3): 230-5, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15735332

ABSTRACT

Glutamate decarboxylase (GAD) is a pyridoxal enzyme that catalyzes the conversion of L-glutamate into gamma-aminobutyric acid and carbon dioxide. The Escherichia coli enzyme exists as two isozymes, referred to as GADalpha and GADbeta. Crystals of the complex of the recombinant isozyme GADalpha with glutarate as a substrate analogue were grown in space group R3, with unit-cell parameters a = b = 117.1, c = 196.4 angstroms. The structure of the enzyme was solved by the molecular-replacement method and refined at 2.05 angstroms resolution to an R factor of 15.1% (R(free) = 19.9%). The asymmetric unit contains a dimer consisting of two subunits of the enzyme related by a noncrystallographic twofold axis which is perpendicular to and intersects a crystallographic threefold axis. The dimers are related by a crystallographic threefold axis to form a hexamer. The active site of each subunit is formed by residues of the large domains of both subunits of the dimer. The coenzyme pyridoxal phosphate (PLP) forms an aldimine bond with Lys276. The glutarate molecule bound in the active site of the enzyme adopts two conformations with equal occupancies. One of the two carboxy groups of the glutarate occupies the same position in both conformations and forms hydrogen bonds with the N atom of the main chain of Phe63 and the side chain of Thr62 of one subunit and the side chains of Asp86 and Asn83 of the adjacent subunit of the dimer. Apparently, it is in this position that the distal carboxy group of the substrate would be bound by the enzyme, thus providing recognition of glutamic acid by the enzyme.


Subject(s)
Escherichia coli/enzymology , Glutamate Decarboxylase/chemistry , Glutarates/chemistry , Glutamate Decarboxylase/isolation & purification , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
10.
Article in English | MEDLINE | ID: mdl-16511092

ABSTRACT

L-Methionine gamma-lyase (MGL) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes gamma-elimination of L-methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9 A resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops.


Subject(s)
Carbon-Sulfur Lyases/chemistry , Citrobacter freundii/enzymology , Bacterial Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Molecular Structure , Protein Structure, Secondary
11.
Mol Biol (Mosk) ; 38(5): 926-36, 2004.
Article in Russian | MEDLINE | ID: mdl-15554194

ABSTRACT

Properties of specific interaction between ribosomal proteins and ribosomal RNAs were analyzed and a method for determination of "recognizing modules" on the protein surface was proposed. The method is based on the search of protein atoms making conserved H-bonds with RNA and forming an invariant spatial structure in homologous rRNA-protein complexes and in the isolated protein. A potential of the method is demonstrated on the determination of the recognizing modules on the surfaces of ribosomal proteins S8, S15 and L5.


Subject(s)
RNA, Ribosomal/metabolism , Ribosomal Proteins/physiology , Bacteria/metabolism , Hydrogen Bonding , Molecular Structure , Mutation/genetics , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism
12.
Biochemistry (Mosc) ; 69(12): 1319-23, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15627386

ABSTRACT

The position and conformation of the N-terminal helix of free ribosomal protein S15 was earlier found to be modified under various conditions. This variability was supposed to provide the recognition by the protein of its specific site on 16S rRNA. To test this hypothesis, we substituted some amino acid residues in this helix and assessed effects of these substitutions on the affinity of the protein for 16S rRNA. The crystal structure of the complex of one of these mutants (Thr3Cys S15) with the 16S rRNA fragment was determined, and a computer model of the complex containing another mutant (Gln8Met S15) was designed. The available and new information was analyzed in detail, and the N-terminal helix was concluded to play no significant role in the specific binding of the S15 protein to its target on 16S rRNA.


Subject(s)
RNA, Ribosomal, 16S/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Crystallization , Models, Molecular , Protein Interaction Mapping , Protein Structure, Secondary , Ribosomal Proteins/genetics , Thermus thermophilus/chemistry , Thermus thermophilus/genetics
13.
Mol Biol (Mosk) ; 35(4): 610-6, 2001.
Article in Russian | MEDLINE | ID: mdl-11524947

ABSTRACT

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilus have earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5-RNA complex crystallized, and its structure determined to 2.3 A. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment-L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.


Subject(s)
RNA, Ribosomal, 5S/metabolism , Ribosomal Proteins/metabolism , Thermus thermophilus/genetics , Thermus thermophilus/metabolism , Binding Sites , Protein Binding , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal, 5S/genetics , Ribosomal Proteins/genetics
14.
Article in Russian | MEDLINE | ID: mdl-11081263

ABSTRACT

50 patients with different neurological manifestations of lumbar osteochondrosis were examined. A new method of transabdominal ultrasonographic investigation of both the discs and the surrounding tissues was applied. 30 cases of discogenic disease and 20 cases of reflex algesic syndrome were revealed. Possibilities of transabdominal ultrasonography for staging the diseases were demonstrated as well as the advantage of the method over computer magnetic-resonance tomography. Ultrasonography allows to visualize rupture of the disc and fibrous changes. The results of the study can be used for diagnosis of the severity of disease as well as for its therapy and prognosis.


Subject(s)
Intervertebral Disc Displacement/diagnostic imaging , Osteochondritis/diagnostic imaging , Reflex/physiology , Cysts/diagnostic imaging , Cysts/pathology , Fibrosis/diagnostic imaging , Fibrosis/pathology , Humans , Lumbosacral Region , Severity of Illness Index , Spinal Diseases/diagnostic imaging , Spinal Diseases/pathology , Spine/diagnostic imaging , Spine/pathology , Syndrome , Ultrasonography
15.
FEBS Lett ; 451(1): 51-5, 1999 May 14.
Article in English | MEDLINE | ID: mdl-10356982

ABSTRACT

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.


Subject(s)
Bacterial Proteins/metabolism , RNA, Ribosomal, 5S/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Thermus thermophilus/metabolism , Amino Acid Sequence , Binding Sites , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Ribosomal, 5S/chemistry , Sequence Homology, Amino Acid
16.
Biol Chem ; 379(7): 795-805, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9705143

ABSTRACT

Crystal and solution structures of fourteen ribosomal proteins from thermophilic bacteria have been determined during the last decade. This paper reviews structural studies of ribosomal proteins from Thermus thermophilus carried out at the Institute of Protein Research (Pushchino, Russia) in collaboration with the University of Lund (Lund, Sweden) and the Center of Structural Biochemistry (Karolinska Institute, Huddinge, Sweden). New experimental data on the crystal structure of the ribosomal protein L30 from T. thermophilus are also included.


Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Bacterial Proteins/metabolism , Binding Sites , RNA/metabolism , Ribosomal Proteins/metabolism
19.
Biochemistry (Mosc) ; 62(2): 221-4, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9159876

ABSTRACT

Ribosomal protein L30 from Thermus thermophilus was overexpressed in E. coli cells. The recombinant protein was isolated and crystallized. The crystals belong to the spatial group P3(1) 12, and their crystallographic parameters are not different from those of crystals obtained earlier from the ribosomal protein isolated from T. thermophilus.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Genes, Bacterial , Ribosomal Proteins/genetics , Ribosomal Proteins/isolation & purification , Thermus thermophilus/genetics , Base Sequence , Crystallization , Escherichia coli/genetics , Gene Expression , Oligonucleotide Probes/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
20.
Proteins ; 27(2): 309-10, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9061793

ABSTRACT

Crystals have been obtained for recombinant ribosomal protein S8 from Thermus thermophilus produced by Escherichia coli. The protein crystals have been grown in 40 mM potassium phosphate buffer (pH 6.0) in hanging drops equilibrated against saturated ammonium sulfate (unbuffered) with 2-methyl-2,4-pentandiol (v/v). The crystals belong to the space group P4(1(3)) 2(1)2 with cell parameters a = b = 67.65 A, c = 171.12 A. They diffract x-rays to 2.9 A resolution.


Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Crystallization , Crystallography, X-Ray , Recombinant Fusion Proteins/chemistry
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