Biomedical subjects

Joachim Behlke

Publications and source records attributed to Joachim Behlke.

At least 19 recordsLinked to original sources

PubMed2006

Self-association of adrenodoxin studied by using analytical ultracentrifugation.

The mitochondrial steroid hydroxylase system of vertebrates utilizes adrenodoxin (Adx), a small iron-sulfur cluster protein of about 14 kDa as an electron carrier between a reductase and cytochrome P450. Although the crystal structure of this protein has been elucidated, the solution structure of Adx was discussed contrary in the literature [I.A. Pikuleva, K. Tesh, M.R. Waterman, Y. Kim, The tertiary structure of full-length bovine adrenodoxin suggests functional dimers, Arch. Biochem. Biophys. 373 (2000) 44-55; D. Beilke, R. Weiss, F. Löhr, P. Pristovsek, F. Hannemann, R. Bernhardt, H. Rüterjans, A new electron mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin, Biochemistry 41 (2002) 7969-7978]. Therefore, it was necessary to study the self-association of this protein by using analytical ultracentrifugation over a larger concentration range. As could be demonstrated in sedimentation velocity experiments, as well as sedimentation equilibrium runs with explicit consideration of thermodynamic non-ideality, the full-length protein (residues 1-128) in the oxidized state resulted in a monomer-dimer equilibrium (K(a) approximately 3 x 10(2) M(-1)). For truncated Adx (1-108), as well as the reduced Adx, the association behavior was strongly reduced. The consequences of this behavior are discussed with respect to the physiological meaning for the Adx system.

Adrenodoxin↗

PubMed2006

Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11.

SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.

Amyloid beta-Protein Precursor↗

PubMed2006

Minigenes encoding N-terminal domains of human cardiac myosin light chain-1 improve heart function of transgenic rats.

In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (K(D)) for the hVLC-1/1-15-actin complex compared with the K(D) value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 muM) correlated positively with significant (P<0.001) improvements of the intrinsic contractile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart.

Actins↗

PubMed2005

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1).

BACKGROUND: Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs). RESULTS: Good quality crystals that were suitable for X-ray crystallographic studies were obtained following the truncation of the 14 N-terminal amino acids of APEG-1, a region predicted to be disordered. The truncated protein (termed DeltaAPEG-1) consists of a single immunoglobulin (Ig) like domain which includes an Arg-Gly-Asp (RGD) adhesion recognition motif. The RGD motif is crucial for the interaction of extracellular proteins and plays a role in cell adhesion. The X-ray structure of DeltaAPEG-1 was determined and was refined to sub-atomic resolution (0.96 A). This is the best resolution for an immunoglobulin domain structure so far. The structure adopts a Greek-key beta-sandwich fold and belongs to the I (intermediate) set of the immunoglobulin superfamily. The residues lying between the beta-sheets form a hydrophobic core. The RGD motif folds into a 310 helix that is involved in the formation of a homodimer in the crystal which is mainly stabilized by salt bridges. Analytical ultracentrifugation studies revealed a moderate dissociation constant of 20 microM at physiological ionic strength, suggesting that APEG-1 dimerisation is only transient in the cell. The binding constant is strongly dependent on ionic strength. CONCLUSION: Our data suggests that the RGD motif might play a role not only in the adhesion of extracellular proteins but also in intracellular protein-protein interactions. However, it remains to be established whether the rather weak dimerisation of APEG-1 involving this motif is physiologically relevant.

Amino Acid Motifs↗

PubMed2005

Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein.

sorLA (Sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid beta-peptide (Abeta), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to Abeta, whereas ablation of sorLA expression in knockout mice results in increased levels of Abeta in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into Abeta and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase Abeta production and plaque formation and promote spontaneous AD.

Alzheimer Disease↗

PubMed2005

Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation.

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-d-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PSIIcc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 +/- 18 kDa and 710 +/- 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 A, which has been improved recently to 3.2 A [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].

Bacterial Proteins↗

PubMed2005

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization.

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.

Amino Acid Sequence↗

PubMed2005

Ahnak is critical for cardiac Ca(V)1.2 calcium channel function and its beta-adrenergic regulation.

Defective L-type Ca2+ channel (I(CaL)) regulation is one major cause for contractile dysfunction in the heart. The I(CaL) is enhanced by sympathetic nervous stimulation: via the activation of beta-adrenergic receptors, PKA phosphorylates the alpha1C(Ca(V)1.2)- and beta2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on I(CaL). Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both beta2 subunit interaction and I(CaL) regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and beta2 subunit decreased by approximately 50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on I(CaL) increasing the amplitude by approximately 60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of I(CaL) by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I(CaL). Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase I(CaL). This genetic ahnak variant might cause individual differences in I(CaL) regulation upon physiological challenges or therapeutic interventions.

Animals↗

PubMed2005

Role of the N-terminal region and of beta-sheet residue Thr29 on the activity of the omega2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035.

The dimeric regulatory protein wild-type omega (wt omega2) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each omega2 protein probably binds with an antiparallel beta-sheet structure in the major groove of the 7-bp subsite of the operator DNA. Exchange of threonine at position 29 to alanine (T29A) drastically affects the activity of variant protein omega2T29A both in vivo and in vitro, and reduces the thermodynamic stability deltaG(o)u, but does not change the conformation. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the omega2T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 microM for wt omega2 to 6.3 microM for omega2T29A. Denatured dimers are formed upon thermal unfolding of wt omega2 and omega2T29A at ca. 45 microM (D(n)<-->D(u)). Removal of 8 (omega2deltaN8), or even 18 (omega2deltaN18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt omega2. The stability of variants omega2deltaN8 and omega2deltaN18 is similar to that of wt omega2, and their binding to operator DNA is not impaired.

Bacterial Proteins↗

PubMed2004

X-ray structure of fumarylacetoacetate hydrolase family member Homo sapiens FLJ36880.

The human protein FLJ36880 belongs to the fumarylacetoacetate hydrolase family. The X-ray structure of FLJ36880 has been determined to 2.2 A resolution employing the semi-automated high-throughput structural genomics approach of the Protein Structure Factory. FLJ36880 adopts a mixed beta-sandwich roll fold and forms homodimers in crystals as well as in solution. One Mg2+ ion is bound to each subunit of the dimeric protein by coordination to three carboxylate oxygens and three water molecules. These metal binding sites are accessible from the same surface of the dimer, partly due to the disorder of the undecapeptide stretch D29 to L39. The overall structure and metal binding site of FLJ36880 bear clear similarities to the C-terminal domain of the bifunctional enzyme HpcE from Escherichia coli C, fumarylacetoacetate hydrolase from Mus musculus and to YcgM (Apc5008) from E. coli 1262. These similarities provide a framework for suggesting biochemical functions and evolutionary relationships of FLJ36880. It appears highly probable that the metal binding sites are involved in an enzymatic activity related to the catabolism of aromatic amino acids. Two point mutations in the active-site of FAH, responsible for the metabolic disease hereditary tyrosinemia type I (HTI) in humans, affect residues that are structurally conserved in FLJ36880 and located in the putative catalytic site.

Amino Acid Sequence↗

PubMed2003

Sedimentation equilibrium: a valuable tool to study homologous and heterogeneous interactions of proteins or proteins and nucleic acids.

We present a short overview of our experience in analyzing the affinity and stoichiometry of self-associating and heterologous interactions by using the sedimentation equilibrium technique. Data acquisition and the fitting procedure employing the computer programs that we have developed, Polymole and Virial, are utilized for obtaining reliable results under ideal as well as non-ideal conditions. Such data derived from biologically important macromolecules find utility in understanding physiological events such as cell regulation.

Algorithms↗

PubMed2003

Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid.

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K(m) ( approximately 0.4 microM) for all variants but slightly different V(max) values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V(max) of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K(m) values of 111, 83, and 250 microM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.

Alleles↗

PubMed2003

Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation.

The IkappaB kinase (IKK) complex mediates activation of transcription factor NF-kappaB by phosphorylation of IkappaB proteins. Its catalytic subunits, IKKalpha and IKKbeta, require association with the regulatory IKKgamma (NEMO) component to gain full basal and inducible kinase activity. However, the oligomeric composition of the IKK complex and its regulation by IKKgamma are poorly understood. We show here that IKKgamma predominantly forms tetramers and interacts with IKKalpha or IKKbeta in this state. We propose that tetramerization is accomplished by a prerequisite dimerization through a C-terminal coiled-coil minimal oligomerization domain (MOD). This is followed by dimerization of the dimers with their N-terminal sequences. Tetrameric IKKgamma sequesters four kinase molecules, yielding a gamma(4)(alpha/beta)(4) stoichiometry. Deletion of the MOD leads to loss of tetramerization and of phosphorylation of IKKbeta and IKKgamma, although the kinase can still interact with the resultant IKKgamma monomers and dimers. Likewise, MOD-mediated IKKgamma tetramerization is required to enhance IKKbeta kinase activity when overexpressed in 293 cells and to reconstitute a lipopolysaccharide-responsive IKK complex in pre-B cells. These data thus suggest that IKKgamma tetramerization enforces a spatial positioning of two kinase dimers to facilitate transautophosphorylation and activation.

Biopolymers↗

PubMed2002

The tailspike protein of Shigella phage Sf6. A structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain.

Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike of Salmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel beta-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel beta-helix protein with high structural similarity to its functional homolog from phage P22.

Amino Acid Sequence↗

PubMed2002

EcoRII: a restriction enzyme evolving recombination functions?

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5'CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two-domain structure that enables this particular mode of protein-DNA interaction. The C-terminal domain is a new restriction endonuclease, EcoRII-C. In contrast to the wild-type enzyme, EcoRII-C cleaves DNA specifically at single 5'CCWGG sites. Moreover, substrates containing two or more cooperative 5'CCWGG sites are cleaved much more efficiently by EcoRII-C than by EcoRII. The N-terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5'CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA-binding domain to enable the interaction with two 5'CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons.

Base Sequence↗

PubMed2002

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.

Binding Sites↗

PubMed2002

CopR binds and bends its target DNA: a footprinting and fluorescence resonance energy transfer study.

Plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. Previous data suggested that CopR is a HTH protein belonging to a family of 578 HTH proteins (termed HTH 3-family). Only a very limited number of proteins in this family, among them lambda c1 repressor, 434 c1 repressor and P22 c2 repressor, have been characterized in detail so far. Previously, a CopR structural model was built based on structural homologies to the 434 c1 and P22 c2 repressor and used to identify amino acids involved in DNA binding and dimerization. Site-directed mutagenesis in combination with electrophoretic mobility shift assay (EMSA), dimerization studies and circular dichroism (CD) measurements verified the model predictions. In this study we used hydroxyl radical footprinting and fluorescence resonance energy transfer (FRET) measurements to obtain detailed information about the structure of the DNA in the CopR-DNA complex. Our results show that the DNA is bent gently around the protein, comparable to the bending angle of 20-25 degrees observed in the 434 c1 repressor-DNA complex and the lambda c1 repressor-DNA complex. The shape of CopR dimers as determined by sedimentation velocity experiments is extended and accounts for the relatively large area of protection observed with hydroxyl radical footprinting.

Bacterial Proteins↗

PubMed2002

A new approximate whole boundary solution of the Lamm differential equation for the analysis of sedimentation velocity experiments.

Sedimentation velocity is one of the best-suited physical methods for determining the size and shape of macromolecular substances or their complexes in the range from 1 to several thousand kDa. The moving boundary in sedimentation velocity runs can be described by the Lamm differential equation. Fitting of suitable model functions or solutions of the Lamm equation to the moving boundary is used to obtain directly sedimentation and diffusion coefficients, thus allowing quick determination of size, shape and other parameters of macromolecules. Here we present a new approximate whole boundary solution of the Lamm equation that simultaneously allows the specification of sedimentation and diffusion coefficients with deviations smaller than 1% from the expected values.

Algorithms↗