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Jürgen Kuhlmann

Publications and source records attributed to Jürgen Kuhlmann.

At least 19 recordsLinked to original sources
PubMed2006

Membrane binding of a lipidated N-Ras protein studied in lipid monolayers.

The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy.

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.

Amino Acid Sequence↗
PubMed2006

Lipidated ras and rab peptides and proteins--synthesis, structure, and function.

Chemical biology can be defined as the study of biological phenomena from a chemical approach. Based on the analysis of relevant biological phenomena and their structural foundation, unsolved problems are identified and tackled through a combination of chemistry and biology. Thus, new synthetic methods and strategies are developed and employed for the construction of compounds that are used to investigate biological procedures. Solid-phase synthesis has emerged as the preferred method for the synthesis of lipidated peptides, which can be chemoselectively ligated to proteins of the Ras superfamily. The generated peptides and proteins have solved biological questions in the field of the Ras-superfamily GTPases that are not amendable to chemical or biological techniques alone.

Animals↗
PubMed2006

Insertion of lipidated Ras proteins into lipid monolayers studied by infrared reflection absorption spectroscopy (IRRAS).

Ras proteins have to be associated with the inner leaflet of the plasma membrane to perform their signaling functions. This membrane targeting and binding is controlled by post-translational covalent attachment of farnesyl and palmitoyl chains to cysteines in the membrane anchor region of the N- and H-Ras isoforms. Two N-Ras lipoproteins were investigated, namely a farnesylated and hexadecylated protein, presenting the natural hydrophobic modifications and a doubly hexadecylated construct, respectively. The proteins are surface active and form a Gibbs monolayer at the air-D2O interface. The contours of the amide-I bands were analyzed using infrared reflection absorption spectroscopy (IRRAS). Langmuir monolayers of a mixture of POPC, brain sphingomyelin, and cholesterol were used as half of a model biomembrane to study the insertion of these N-Ras proteins. They insert with their hydrophobic anchors into lipid monolayers but at higher surface pressures (30 mN/m); the farnesylated and hexadecylated protein desorbs completely from the monolayer, whereas the doubly hexadecylated protein remains incorporated. During the insertion process, changes in the orientation of the protein secondary structure were detected by comparison with simulated IRRA spectra, based on the information on the relative orientation of the secondary structure elements from the protein crystal structure data.

Adsorption↗
PubMed2006

A generic building block for C- and N-terminal protein-labeling and protein-immobilization.

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.

Azides↗
PubMed2006

The alpha-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit.

Chloroplasts of higher plants contain a unique signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. CpSRP43 is composed of a four ankyrin repeat domain and three functionally distinct chromodomains (CDs). In this report we confirm previously published data that the second chromodomain (CD2) provides the primary binding site for cpSRP54. However, quantitative binding analysis demonstrates that cpSRP54 binds to CD2 significantly less efficiently than it binds to full-length cpSRP43. Further analysis of the binding interface of cpSRP by mutagenesis studies and a pepscan approach demonstrates that the C-terminal alpha-helix of CD2 facilitates binding to cpSRP54.

Amino Acid Sequence↗
PubMed2006

Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption.

In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) >> s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.

Boron Compounds↗
PubMed2006

Influence of serine O-glycosylation or O-phosphorylation close to the vJun nuclear localisation sequence on nuclear import.

Nuclear import triggered by the nuclear-localisation sequence (NLS) of the viral Jun (vJun) protein is mediated by phosphorylation of a serine close to the NLS. Since phosphorylation and glycosylation of serine residues are often in a reciprocal "yin-yang" relationship, we investigated whether glycosylation of this serine with O-linked N-acetylglucosamine (O-GlcNAc) would also regulate nuclear import via the vJun NLS. Peptides containing the vJun NLS with an adjacent O-phosphorylated, O-GlcNAc-functionalised or unmodified serine, and equipped with an N-terminal biotin or a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent label, were synthesised on the solid phase by means of an Fmoc/Boc strategy and a Pd0-sensitive HYCRON linker. Fluorescence-polarisation measurements on the NBD-labelled peptides indicated that modification with phosphate or O-GlcNAc leads to a decrease in affinity to the import-mediating adapter protein, importin alpha, of about one order of magnitude compared to the unmodified NLS. Microinjection of biotinylated NLS peptide conjugated with fluorescently labelled avidin into NIH/3T3 and MDCK cells, revealed that avidin-unmodified-NLS peptide was rapidly imported into the nucleus. However, either phosphate or O-GlcNAc next to the NLS caused almost complete exclusion of the protein conjugate from nuclear import. These findings indicate that nuclear import by the vJun NLS might not be regulated by a "yin-yang" modification of an adjacent serine with phosphate or O-GlcNAc. Rather, negative regulation of binding between the polybasic NLS and importin by a negatively charged or a bulky, uncharged residue appears likely.

Active Transport, Cell Nucleus↗
PubMed2005

In deuterated water the unspecific adsorption of proteins is significantly slowed down: results of an SPR study using model organic surfaces.

The control of unspecific adsorption of proteins to natural and technical surfaces plays an important role in biology and also for many applications. Organic model surfaces, e.g., self-assembled monolayers, are often used to identify fundamental surface and/or protein properties that rule protein adsorption. Some techniques involved in biointerface research require the use of heavy water, e.g. neutron scattering techniques. Also in NMR studies D(2)O is the solvent of choice when focusing on biomolecular and hydration dynamics. So far several studies have been concerned with the characterization of the unspecific adsorption of proteins from normal water buffers. In the present work, we report a comparison of the unspecific protein adsorption from normal and heavy water buffers. So far it has been assumed that the surface kinetic of the unspecific adsorption is unaffected by the substitution of water by D(2)O. However, for the four proteins investigated here, this assumption does not hold. The ratio k(H)/k(D) of the adsorption rate constants of the different buffer conditions describe the strength of the isotope effect. We have measured ratios between 1.0 and 2.6, indicating that the adsorption kinetics are strongly affected by a H(2)O-D(2)O substitution.

Adsorption↗
PubMed2005

Solution structure of the Ran-binding domain 2 of RanBP2 and its interaction with the C terminus of Ran.

The termination of export processes from the nucleus to the cytoplasm in higher eukaryotes is mediated by binding of the small GTPase Ran as part of the export complexes to the Ran-binding domains (RanBD) of Ran-binding protein 2 (RanBP2) of the nuclear pore complex. So far, the structures of the first RanBD of RanBP2 and of RanBP1 in complexes with Ran have been known from X-ray crystallographic studies. Here we report the NMR solution structure of the uncomplexed second RanBD of RanBP2. The structure shows a pleckstrin homology (PH) fold featuring two almost orthogonal beta-sheets consisting of three and four strands and an alpha-helix sitting on top. This is in contrast to the RanBD in the crystal structure complexes in which one beta-strand is missing. That is probably due to the binding of the C-terminal alpha-helix of Ran to the RanBD in these complexes. To analyze the interaction between RanBD2 and the C terminus of Ran, NMR-titration studies with peptides comprising the six or 28 C-terminal residues of Ran were performed. While the six-residue peptide alone does not bind to RanBD2 in a specific manner, the 28-residue peptide, including the entire C-terminal helix of Ran, binds to RanBD2 in a manner analogous to the crystal structures. By solving the solution structure of the 28mer peptide alone, we confirmed that it adopts a stable alpha-helical structure like in native Ran and therefore serves as a valid model of the Ran C terminus. These results support current models that assume recognition of the transport complexes by the RanBDs through the Ran C terminus that is exposed in these complexes.

Models, Molecular↗
PubMed2005

Double-wavelength technique for surface plasmon resonance measurements: basic concept and applications for single sensors and two-dimensional sensor arrays.

A new technique for on-line monitoring of analyte binding to sensor surfaces by surface plasmon resonance (SPR) detection is described. It is based on differential measurements using two wavelengths provided by two diode lasers. The technique is as simple and robust as the conventional SPR detection measuring the reflected radiation at fixed incidence angle, but it has the advantage of being nonsensitive to variations of the resonance width and providing essentially higher signal/noise ratios. The paper presents the first four channel prototype system for parallel 2D-monitoring at four different spots. One channel is always used as a reference to compensate temperature fluctuations and nonspecific adsorptions. Calibration with sucrose solutions revealed an absolute sensitivity of Deltan approximately 5 x 10(-6). The new technique is tested with a biotin-streptavidin binding and with hybridization/denaturation of DNA. Biotin binding to a streptavidin monolayer is detected with a signal/noise ratio of about 5, which demonstrates the high potential of the new technique for applications in drug discovery. Applications to gene analysis are tested with short oligonucleotides of the sequences used for genotyping human hepatitis C viruses. A selective response to complementary oligonucleotides is observed. The high reproducibility in subsequent cycles of hybridization/denaturation (by formamide or by heating) points out potential applications of the technique in medical diagnostics, food industry, genomics, and proteomics too.

Biosensing Techniques↗
PubMed2005

The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints.

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.

Air Pollutants↗
PubMed2005

An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

Acylation↗
PubMed2005

Synthesis and application of fluorescent ras proteins for live-cell imaging.

Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.

Amino Acid Sequence↗
PubMed2004

The cholesterol membrane anchor of the Hedgehog protein confers stable membrane association to lipid-modified proteins.

The Hedgehog proteins are potent organizers of animal development. They carry a cholesterol ester at the C terminus of their signaling domain. The membrane anchoring mediated by this lipophilic modification was studied by means of an approach integrating cell biology, biochemistry, biophysics, and organic chemistry techniques. Sterol-modified and fluorescent-labeled Hedgehog-derived peptides and proteins were synthesized and investigated in biophysical and cell-biological assays. These experiments revealed that cholesterol alone anchors proteins to membranes with significant strength and half-times for spontaneous desorption of several hours. Its membrane anchoring ability is comparable to dual lipidation motifs such as double geranylgeranylation or S-palmitoylation plus S-farnesylation found in other lipidated proteins. The experiments also demonstrate that membrane binding changes dramatically if short lipidated peptides are equipped with a large protein. These data suggest that for Hedgehog release and subsequent signaling an interaction partner such as the Dispatched protein is necessary. In addition to these findings the described approach allows one to correlate biophysical data obtained with model peptides with data determined with fully functional proteins and to combine results from in vitro and in vivo experiments. It should be generally applicable to other membrane anchors and proteins.

Animals↗