Biomedical subjects

Helena Tossavainen

Publications and source records attributed to Helena Tossavainen.

9 recordsLinked to original sources

PubMed2006

Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments.

Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a powerful strategy for the assignment of methyl-containing residues in a uniformly 13C/15N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions, the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity. A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours.

Amino Acid Sequence↗

PubMed2006

Solution structures of the first and fourth TSR domains of F-spondin.

F-spondin is a protein mainly associated with neuronal development. It attaches to the extracellular matrix and acts in the axon guidance of the developing nervous system. F-spondin consists of eight domains, six of which are TSR domains. The TSR domain family binds a wide range of targets. Here we present the NMR solution structures of TSR1 and TSR4. TSR domains have an unusual fold that is characterized by a long, nonglobular shape, consisting of two beta-strands and one irregular extended strand. Three disulfide bridges and stack of alternating tryptophan and arginine side-chains stabilize the structure. TSR1 and TSR4 structures are similar to each other and to the previously determined TSR domain X-ray structures from another protein, TSP, although TSR4 exhibits a mobile loop not seen in other structures.

Amino Acid Sequence↗

PubMed2006

Peptide binding and NMR analysis of the interaction between SAP97 PDZ2 and GluR-A: potential involvement of a disulfide bond.

Synaptic delivery of GluR-A (GluR1) subunit-containing glutamate receptors depends on a C-terminal type I PDZ binding motif in GluR-A. Synapse-associated protein 97 (SAP97) is the only PDZ domain protein known to associate with GluR-A. We have used NMR spectroscopy and a biotinylated peptide binding assay to characterize the interaction between synthetic GluR-A C-terminal peptides and the PDZ2 domain of SAP97 (SAP97(PDZ2)), previously determined to be the dominant factor responsible for the interaction. The binding mode appeared to be strongly influenced by redox conditions. Chemical shift changes observed in NMR spectra indicate that under reducing conditions, the last four residues of GluR-A peptides bind to PDZ2 in a fashion typical of class I PDZ interactions. The binding is weak and relatively nonselective as it occurs similarly with a PDZ2 domain derived from PSD-95, a related protein not believed to directly interact with GluR-A. In the absence of reducing agents, conserved cysteine residues in SAP97(PDZ2) and the GluR-A C-terminus gave rise to an anomalous behavior in a microplate assay with a biotinylated GluR-A 18-mer peptide. A covalent disulfide-linked complex between SAP97(PDZ2) and the GluR-A peptide was seen in the binding assay and in the NMR experiments performed under oxidizing conditions. The results are consistent with a two-step binding mechanism consisting of an initial PDZ interaction followed by stabilization of the complex by a disulfide bond. The possible physiological relevance of redox regulation of SAP97-GluR-A interaction remains to be established.

Adaptor Proteins, Signal Transducing↗

PubMed2006

NMR solution structure and characterization of substrate binding site of the PPIase domain of PrsA protein from Bacillus subtilis.

PrsA is a peptidyl-prolyl isomerase (PPIase) from Bacillus subtilis belonging to the parvulin family of PPIases. It is a membrane bound lipoprotein at the membrane-wall interface, involved in folding of exported proteins. We present the NMR solution structure of the PPIase domain of PrsA, the first from a Gram-positive bacterium. In addition we mapped out the active site with NMR titration experiments. A high degree of conservation with other members of the parvulin family was revealed in the structure and binding site. Interactions with substrate peptides were also characterized by mutated domains revealing that H122 is indispensable for overall correct folding.

Bacillus subtilis↗

PubMed2006

Simultaneous determination of one- and two-bond scalar and residual dipolar couplings between 13C', 13Calpha and 15N spins in proteins.

Four simple and sensitive HNCO-based methods for measurement of 1J(C'Calpha), 1J(NCalpha) and 2J(NCalpha) coupling constants in protein main chains are presented. Three of these experiments enable the simultaneous measurement of 1J(C'Calpha), 1J(NCalpha) and 2J(NCalpha) couplings. Exploitation of the E.COSY principle provides excellent dispersion of cross peaks in the resulting 3D spectra. The couplings can be retrieved with good accuracy from peak-to-peak separations. Karplus parameterizations are provided for 1J(NCalpha) and 2J(NCalpha), obtained from a nearly complete set of couplings of human ubiquitin. In addition, feasibility of the proposed methodology for measuring several residual dipolar couplings (RDCs) simultaneously is assessed.

Carbon Isotopes↗

PubMed2006

The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site.

Thrombospondin-related anonymous protein, TRAP, has a critical role in the hepatocyte invasion step of Plasmodium sporozoites, the transmissible form of the parasite causing malaria. The extracellular domains of this sporozoite surface protein interact with hepatocyte surface receptors whereas its intracellular domain acts as a link to the sporozoite actomyosin motor system. Liver heparan sulfate proteoglycans have been identified as potential ligands for TRAP. Proteoglycan binding has been associated with the A- and TSR domains of TRAP. We present the solution NMR structure of the TSR domain of TRAP and a chemical shift mapping study of its heparin binding epitope. The domain has an elongated structure stabilized by an array of tryptophan and arginine residues as well as disulfide bonds. The fold is very similar to those of thrombospondin type-1 (TSP-1) and F-spondin TSRs. The heparin binding site of TRAP-TSR is located in the N-terminal half of the structure, the layered side chains forming an integral part of the site. The smallest heparin fragment capable of binding to TRAP-TSR is a tetrasaccharide.

Animals↗

PubMed2004

Efficient assignment of methyl resonances: enhanced sensitivity by gradient selection in a DE-MQ-(H)CC(m)Ht (m)-TOCSY experiment.

We present a gradient selected and doubly sensitivity-enhanced DE-MQ-(H)CC(m)H(m)-TOCSY experiment for the sequence-specific assignment of methyl resonances in (13)C,(15)N labeled proteins. The proposed experiment provides improved sensitivity and artifact suppression relative to the phase-cycled experiments. One part of the (13)Cchemical shift evolution takes place under heteronuclear multiple quantum coherence, whereas the other part occurs under (13)C single quantum coherence in a semi-constant time fashion. The feasibility of the experiment was assessed using (15)N,(13)C labeled Mus musculus coactosin (16 kDa), having a rotational correlation time of 14.5 ns at 15 degrees C in D(2)O. A 16-h experiment on 600 MHz (1)H yielded good quality data and enabled the assignment of 70 out of 72 methyl groups in coactosin. As well as being an improved approach for methyl resonance assignment, this experiment can also be highly valuable for the rapid assignment of methyl resonances in SAR by NMR studies.

Animals↗

PubMed2004

Optimized pathway selection in intraresidual triple-resonance experiments.

An optimized intraresidual pulse sequence element with better sensitivity and suppression of sequential cross peaks is presented. Concatenation of three magnetization transfer delays allows their independent setting, in accordance with the relaxation properties of the individual spins, without concomitantly prolonging the pulse sequence. Additionally, implementations of the scheme to HNCA, HNCACB, and the TROSY based triple-resonance experiments are proposed. The feasibility of the new element was verified by recording HNCA and HNCACB on the small 8.6 kDa protein ubiquitin. The corresponding HNCA-TROSY experiment was tested on a larger protein, the 30.4 kDa Cel6A from the thermophilic soil bacterium Thermobifida fusca at 800 (1)H MHz.

Carbon Isotopes↗

PubMed2003

NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.

Amino Acid Sequence↗