Biomedical subjects

Perttu Permi

Publications and source records attributed to Perttu Permi.

26 records · Page 2Linked to original sources

PubMed2003

The peptide-substrate-binding domain of human collagen prolyl 4-hydroxylases. Backbone assignments, secondary structure, and binding of proline-rich peptides.

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are alpha 2 beta 2 tetramers in which protein-disulfide isomerase serves as the beta subunit. Two isoforms of the catalytic alpha subunit have been identified and shown to form [alpha(I)]2 beta 2 and [alpha(II)]2 beta 2 tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue alpha(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H alpha(I) polypeptide Phe144-Ser244 forms a folded domain consisting of five alpha helices and one short beta strand. This structure is quite different from those of other proline-rich peptide-binding modules, which consist mainly of beta strands. Binding of the peptide (Pro-Pro-Gly)2 to this domain caused major chemical shifts in many backbone amide resonances, the residues showing the largest shifts being mainly hydrophobic, including three tyrosines. The Kd values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the alpha(I) and the corresponding alpha(II) domain were very similar to the Km and Ki values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers. The Kd values of the alpha(I) and alpha(II) domains for (Gly-Pro-4Hyp)5 were much higher than those for (Pro-Pro-Gly)5, indicating a marked decrease in the affinity of hydroxylated peptides for the domain. Many characteristic features of the binding of peptides to the type I and type II C-P4H tetramers can thus be explained by the properties of binding to this domain rather than the catalytic domain.

Amino Acid Sequence↗

PubMed2003

Determination of three-bond scalar coupling between 13C' and 1H(alpha) in proteins using an iHN(CA),CO(alpha/beta-J-COHA) experiment.

Triple-resonance NMR experiments for measuring three-bond scalar coupling constant between 13C' (i-1) and 1H(alpha)(i) spins, defining the dihedral angle phi, are presented. The novel experiments enable the measurement of 3JC'H(alpha)) from simple two (or three)-dimensional 13C', (15N/13C(alpha)), 1H(N) correlation spectra with minimal resonance overlap, thanks to solely intraresidual coherence transfer pathway and spin-state-selection. The 3J(C'H(alpha)) values measured in human ubiquitin using the proposed intraresidual iHN(CA),CO(alpha/beta-J-COHA) TROSY method were compared with those determined previously utilizing the HCAN[C'] experiment.

Algorithms↗

PubMed2003

Measurement of residual dipolar couplings from 1Halpha to 13Calpha and 15N using a simple HNCA-based experiment.

Novel NMR pulse schemes for simultaneous measurement of 1D(C)alpha(H)alpha and 2D(NH)alpha residual dipolar couplings in proteins is presented. We show that 2D(NH)alpha coupling can be very useful for protein structure determination. The 2D(NH)alpha coupling can be measured from 15N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large 1D(C)alpha(H)alpha coupling. The dynamic range of 2D(NH)alpha coupling is comparable to 1D(NC') coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on 15N/13C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both 1D(C)alpha(H)alpha and 2D(NH)alpha couplings, was obtained.

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↗

PubMed2002

Evaluation and optimization of coherence transfer in high molecular weight systems.

For very large proteins in the highest magnetic fields, the large chemical shift anisotropy (CSA) of carbonyl carbon deteriorates coherence transfer efficiency in experiments designed for unambiguous sequential backbone assignment. In this communication, coherence throughput of several TROSY experiments is evaluated. Two new experiments, MP-HNCA and HN(CO)CANH, are also introduced as attractive alternatives for sequential assignment purposes of large proteins with correlation time over 50 ns. Their theoretical coherence transfer efficiencies for the interresidual (13)C(alpha) correlations are significantly better than in recently introduced MP-CT-HNCA and sequential HNCA experiments. The improvement with the new experiments is observed already on 60.8 kDa homodimer of protein Cel6A at 800 (1)H MHz.

Carbon Isotopes↗

PubMed2002

A spin-state-selective experiment for measuring heteronuclear one-bond and homonuclear two-bond couplings from an HSQC-type spectrum.

Recently, a set of selective 1D experiments with spin-state-selective excitation for CH spin systems was introduced by Parella and Belloc (J. Magn. Reson., 148, 78-87 (2001)). We have expanded and generalized this concept further, and demonstrated that a very simple experiment utilizing spin-state-selective filtering can be used for simultaneous measurement of heteronuclear 1JNH (or 1JCH) and geminal 2JHH couplings from two-dimensional 15N-1H (or 13C-1H) correlation spectrum. The experiment has very high sensitivity owing to the preservation of equivalent coherence transfer pathways analogous to the sensitivity and gradient enhanced HSQC experiment. However, overall length of the pulse sequence is 1/(2J) shorter than the gradient selected SE-HSQC experiment. Furthermore, the spin-state-selection can be utilized between NH and NH2 (or CH and CH2) moieties by changing the phase of only one pulse. The pulse scheme will be useful for the measurement of scalar and residual dipolar couplings in wide variety of samples, due to its high sensitivity and artifact suppression efficiency. The method is tested on NH2 and CH2 moieties in 15N- and 15N/13C-labeled ubiquitin samples.

Carbon Isotopes↗

PubMed2002

Intraresidual HNCA: an experiment for correlating only intraresidual backbone resonances.

Resonance overlap in 13Calpha-dimension can seriously deteriorate sequential assignment of proteins, especially in the case of highly alpha helical or partially unfolded structures. In this paper, two novel triple-resonance experiments, for obtaining solely intraresidual HN, N, Calpha correlations, are introduced. The proposed experiments are complementary to the conventional HN(CO)CA experiment, and can be utilized for the sequential assignment of 15N/13C/(2H)-labeled proteins. Coherence transfer efficiency of the new experiment is comparable to the conventional HNCA experiment on proteins with sufficiently long 15N transverse relaxation time. These new coherence transfer schemes are also very useful building blocks for experiments gathering structural information, such as J-couplings, exclusively on the intraresidual alpha carbon. Experimental assessment is demonstrated on ubiquitin at 600 1H MHz.

Animals↗

PubMedDate not supplied

Radionuclide imaging of tumor xenografts in mice using a gelatinase-targeting peptide.

Tumors express MMP-2 and MMP-9 gelatinases, which are involved in the formation of tumor vasculature. This suggests that a tumor and its surrounding neovasculature can be visualized by a sensitive gelatinase recognition method. We have studied tumor radioimaging using a gelatinase inhibitory peptide CTTHWGFTLC (CTT), which in a mouse model targets the tumor site following an intravenous injection. We determined a solution NMR structure of CTT and its retro-inversion peptide, and prepared 125I and 99mTc-labelled CTT peptide derivatives. Radiolabelled CTT inhibited gelatinases in vitro, and homed to a tumor xenograft in mice. In normal mice, CTT was instead rapidly cleared from the circulation mainly through the kidney and, after 24 h, no significant radioactivity was accumulated in healthy tissues. Gamma camera imaging of a primary tumor in live mice was obtained with double-labelled liposomes, which were coated with 99mTc-CTT and encapsulated with 125I albumin. CTT also targeted liposomes to the lungs of tumor-bearing mice, which may indicate the existence of non-visible lung micrometastases. Our studies suggest that selective gelatinase-targeting compounds could be useful in the early detection and imaging of primary tumors and metastases.

Animals↗