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M Kjeldgaard

Publications and source records attributed to M Kjeldgaard.

31 records · Page 2Linked to original sources
PubMed1994

Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe-tRNA(Phe).

Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.

Chromatography, Gel↗
PubMed1993

The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation.

BACKGROUND: Elongation factor Tu (EF-Tu) is a GTP-binding protein that is crucial for protein biosynthesis. In the GTP form of the molecule, EF-Tu binds tightly to aminoacyl-tRNA, forming a ternary complex that interacts with the ribosomal acceptor site. During this interaction, GTP is hydrolyzed, and EF-Tu.GDP is ejected. RESULTS: The crystal structure of EF-Tu from Thermus aquaticus, complexed to the GTP analogue GDPNP, has been determined at 2.5 A resolution and compared to the structure of Escherichia coli EF-Tu.GDP. During the transition from the GDP (inactive) to the GTP (active) form, domain 1, containing the GTP-binding site, undergoes internal conformational changes similar to those observed in ras-p21. In addition, a dramatic rearrangement of domains is observed, corresponding to a rotation of 90.8 degrees of domain 1 relative to domains 2 and 3. Residues that are affected in the binding of aminoacyl-tRNA are found in or near the cleft formed by the domain interface. CONCLUSION: GTP binding by EF-Tu leads to dramatic conformational changes which expose the tRNA binding site. It appears that tRNA binding to EF-Tu induces a further conformational change, which may affect the GTPase activity.

Amino Acid Sequence↗
PubMed1992

Refined structure of elongation factor EF-Tu from Escherichia coli.

The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution. In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles. The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels. The GDP-binding domain has a folding that is found in other GDP-binding proteins. Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.

Amino Acid Sequence↗
PubMed1991

GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites.

GTPase domains are functional and structural units employed as molecular switches in a variety of important cellular functions, such as growth control, protein biosynthesis, and membrane traffic. Amino acid sequences of more than 100 members of different subfamilies are known, but crystal structures of only mammalian ras p21 and bacterial elongation factor Tu have been determined. After optimal superposition of these remarkably similar structures, careful multiple sequence alignment, and calculation of residue-residue interactions, we analyzed the two subfamilies in terms of structural conservation, sequence conservation, and residue contact strength. There are three main results. (i) A structure-based alignment of p21 and elongation factor Tu. (ii) The definition of a common conserved structural core that may be useful as the basis of model building by homology of the three-dimensional structure of any GTPase domain. (iii) Identification of sequence regions, other than the effector loop and the nucleotide binding site, that may be involved in the functional cycle: they are loop L4, known to change conformation after GTP hydrolysis; helix alpha 2, especially Arg-73 and Met-67 in ras p21; loops L8 and L10, including ras p21 Arg-123, Lys-147, and Leu-120; and residues located spatially near the N and C termini. These regions are candidate sites for interaction either with the GTP/GDP exchange factor, with a GTPase-affected function, or with a molecule delivered to a destination site with the aid of the GTPase domain.

Amino Acid Sequence↗
PubMed1991

Improved methods for building protein models in electron density maps and the location of errors in these models.

Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.

Chemical Phenomena↗
PubMed1990

Structural determination of the functional sites of E. coli elongation factor Tu.

Recently, we have made significant progress in solving the structure of a nicked form of elongation factor (EF)-Tu complexed with GDP. The structure has been refined to an R factor of 19.2% at 2.6 A resolution, so that most of the structure is clearly visible in the electron density map. Here we describe what is known about functional sites of EF-Tu in terms of the structure, which still lacks amino acids 40-60.

Anti-Bacterial Agents↗
PubMed1988

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli.

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.

Escherichia coli↗
PubMed1987

A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

Bacterial Proteins↗
PubMed1987

The protein synthesis initiation factor 2 G-domain. Study of a functionally active C-terminal 65-kilodalton fragment of IF2 from Escherichia coli.

Protein synthesis initiation factor 2 (IF2) is present in Escherichia coli cells as two forms which are expressed from the same gene: IF2 alpha [97.3 kilodaltons (kDa)] and IF2 beta (79.7 kDa). During isolation, a smaller form, IF2 gamma, is generated, presumably by partial proteolysis. It has been purified to homogeneity and has an apparent mass of 70 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectrophoresis of IF2 alpha and IF2 gamma shows that IF2 gamma is immunologically partially identical with IF2 alpha. The sequence of the 15 N-terminal amino acid residues of IF2 gamma was determined and compared with that of IF2 alpha. The N-terminal amino acid of IF2 gamma corresponds to Arg-290 of IF2 alpha, suggesting that IF2 gamma is generated by proteolytic cleavage of the Lys-289-Arg-290 bond of IF2. Assuming a C terminus identical with IF2 alpha, we calculate that IF2 gamma comprises 601 amino acid residues and has a mass of 64.8 kDa. The truncated protein was tested for activities characteristic of IF2 in three in vitro assays: fMet-tRNA(fMet) binding to 70S ribosomes, N-terminal dipeptide synthesis in a DNA-dependent transcription/translation system, and ribosome-dependent GTP hydroly97-7. The specific activities of IF2 gamma were comparable with, or only slightly less than, those for IF2 alpha, indicating that IF2 gamma contains the active centers for interaction with fMet-tRNA(fMet), ribosomes, and GTP. A central region in the primary structure of IF2 shows extensive sequence homology with a number of GDP-binding proteins and especially with the G-domain of elongation factor Tu (EF-Tu).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

A model for the tertiary structure of p21, the product of the ras oncogene.

A model was developed for the structure of p21, the protein with a molecular weight of 21,000 that is produced by the ras genes. This model predicts that p21 consists of a central core of beta-sheet structure, connected by loops and alpha helices. Four of these loops comprise the guanine nucleotide binding site. The phosphoryl binding region is made up of amino acid sequences from 10 to 16 and from 57 to 63 of p21. The latter sequence may contain a site for magnesium binding. Amino acids defining guanine specificity are Asn-116 and Asp-119, and sequences around amino acid 145 may contribute to guanine binding. The model makes it possible to visualize how oncogenic mutations of p21 affect interaction with guanine nucleotides.

Amino Acids↗
PubMed1984

Trypanosome variant-specific glycoproteins: a polygene protein family with multiple folding patterns?

Infection with the African trypanosomes gives rise to relapsing waves of parasitemia in the host. A predominant population of trypanosomes is present in each wave, and such predominant populations are usually serologically distinct from each other. Trypanosomes are covered by an extramembranous, highly antigenic, variant-specific glycoprotein coat that is 15 nm thick. The primary structure of a large portion of the glycoprotein molecule is different in the predominant trypanosome populations of each parasitemic wave. Analysis of the secondary structure potential of five full-length and five partial amino acid sequences of variant-specific glycoproteins from members of the Trypanosoma brucei complex has been carried out. The potentials for alpha-helix, beta-turns, and beta-strand structure have been calculated. A high degree of alpha-helical structure potential is present in all the full-length or partial sequences examined. There is conservation of secondary structure potential in the COOH-terminal 100 amino acids, where both partial and complete conservation of primary amino acid sequence exists. The NH2-terminal regions are rich in alpha-helix potential. However, over large stretches of the middle of the VSG molecules there is wide diversity of secondary structure potential. This suggests that tertiary folding structures may also be different in this region. If these predictions are true, different regions of the variant-specific glycoprotein could be exposed to the solvent in different variant-specific trypanosome serotypes. The implication is that antigenic variation is mediated by a polygene family of glycoproteins containing highly polymorphic regions. These could fold differently and expose different surface regions of the protein to the solvent. This device might reduce immune crossreactivity among members of the variant-specific glycoprotein family.

Amino Acid Sequence↗