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Perttu Permi

Publications and source records attributed to Perttu Permi.

At least 19 recordsLinked to original sources
PubMed2006

Effect of protein structure on laccase-catalyzed protein oligomerization.

Laccase-catalyzed oligomerization of proteins was studied using Trametes hirsuta laccase (ThL) and coactosin as a model system. The reaction mechanism was elucidated using free amino acids and the tripeptide Gly-Leu-Tyr as substrates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) as well as oxygen consumption measurements and SDS-PAGE were used to study the reactions. Of the 15 selected amino acids, ThL was found to oxidize tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys), of which the reactions with Tyr and Cys have been described earlier. ThL was able to link four full-length coactosins, whereas coactosin that was truncated from its C-terminus remained unpolymerized. Of the four tyrosine residues present in coactosin, only the tyrosine in the C-terminus was found to be reactive. Polymerization between tyrosine side-chains was unambiguously shown using different oligomers of Gly-Leu-Tyr as parent ions in MALDI-TOF/TOF MS fragment ion analyses.

Basidiomycota↗
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↗
PubMed2005

The structure of the GPIb-filamin A complex.

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.

Contractile Proteins↗
PubMed2005

A set of HA-detected experiments for measuring scalar and residual dipolar couplings.

A new set of HCACO based three-dimensional NMR experiments for measuring residual dipolar couplings in proteins is presented. Using spin-state selection and editing in three dimensions, the experiments allow accurate measurement of intraresidual (1)D(C'H(alpha)), (1)D(C'C(alpha)) and (2)D(C'H(alpha)) scalar and residual dipolar couplings of (15) N/(13)C labeled proteins in D(2)O and dilute liquid crystals with minimal spectral crowding. The presented experiments are especially suitable for small or medium sized proline-rich proteins, or proteins that require high pH solvent conditions, making (1)H(N) detected experiments unattractive. In addition, the tetrahedral coordination of C(alpha) is superior to the planar peptide bond for determination of local alignments in partially structured polypeptides. For the efficient use of spectrometer time and to avoid complications arising from the varying magnitude of the alignment tensor during relatively long experiments, the (1)D(C'H(alpha)) and (2)D(C'H(alpha)) couplings can also be measured simultaneously in an E.COSY like manner with high accuracy. The pulse sequences are balanced for cross-correlation effects and minimized for relaxation losses. The pulse sequences are tested with a sample of (15)N/(13)C human ubiquitin. We find internuclear vector directions determined from the dipolar couplings to have an excellent correlation with those of ubiquitin's refined solution structure.

Carbon↗
PubMed2004

Solution structure of coactosin reveals structural homology to ADF/cofilin family proteins.

Coactosin is a small (MW approximately 15 kDa) evolutionarily conserved actin filament binding protein. It displays remote sequence homology to ADF/cofilin proteins and to the ADF-H domains of twinfilin and Abp1/drebrin. However, biochemical analyses have demonstrated that coactosin has a very different role in actin dynamics from the ones of ADF/cofilin, twinfilin or Abp1/drebrin. To elucidate the molecular mechanism of coactosin/actin interaction, we determined the three-dimensional structure of mouse coactosin by multidimensional NMR spectroscopy. We find that the coactosin structure is homologous to ADF/cofilin and to the ADF-H domains of twinfilin. Furthermore, the regions that have been shown to be important for actin filament interactions in ADF/cofilins are structurally conserved in coactosin suggesting that these two proteins interact with F-actin through a conserved interface. Our analysis also identifies key structural differences between these proteins that may account for the differences in biochemical activities and cellular roles of these proteins.

Actin Depolymerizing Factors↗
PubMed2004

On the interpretation of residual dipolar couplings as reporters of molecular dynamics.

The analysis of residual dipolar couplings from an ensemble of conformations to extract molecular dynamics is intricate. The very mechanism that is necessary to perturb overall molecular tumbling to generate nonvanishing residual dipolar couplings gives rise to convoluted data. The measured values are essentially weighted averages over conformations. However, the weights are not simply the populations of conformations. Consequently, the observed order parameter is not exactly the true measure of motion. In the case of paramagnetic alignment, the apparent order parameter is expected to depend on the number of torsions that separate the locus of interest from the paramagnetic site. In the case of alignment due to steric obstruction, the uneven selection of conformations by their differing Saupe order matrices leads to a bias in the residual dipolar couplings-probed molecular dynamics.

Models, Molecular↗
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↗
PubMed2004

Alignment of chain-like molecules.

The steric obstruction model, that describes the enhanced alignment of folded proteins by anisotropic medium, is extended to account for the residual dipolar couplings of chain-like polypeptides. The average alignment of each chain segment is calculated from an ensemble of conformations represented by a spatial probability distribution. The segmental alignment depends on chain length, flexibility and segment's position in the chain. Residual dipolar couplings in turn depend on internuclear vector directions within each fragment. The results of calculations and simulations explain salient features of the experimental data. With this insight residual dipolar couplings can be interpreted to assess the degree of denaturation, local structures and spatial organization of weakly structured proteins.

Data Interpretation, Statistical↗
PubMed2003

On the origin of residual dipolar couplings from denatured proteins.

Effects of steric obstruction on random flight chains are examined. Spatial probability distributions are elaborated to calculate residual dipolar couplings and residual chemical shift anisotropy, parameters that are acquired by NMR spectroscopy from solutes dissolved in dilute liquid crystals. Calculations yield chain length and residue position-dependent values in good agreement with simulations to provide understanding of recently acquired data from denatured proteins.

Anisotropy↗