Biomedical subjects

H Sterk

Publications and source records attributed to H Sterk.

10 recordsLinked to original sources

PubMed2001

Solution structure of the DNA-binding domain of TraM.

The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented. The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy. It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical. The very first amino acids of the N-terminus are unstructured.

Bacterial Proteins↗

PubMed2001

Pure-phase selective excitation in fast-relaxing systems.

Selective pulses have been used frequently for small molecules. However, their application to proteins and other macromolecules has been limited. The long duration of shaped-selective pulses and the short T(2) relaxation times in proteins often prohibited the use of highly selective pulses especially on larger biomolecules. A very selective excitation can be obtained within a short time by using the selective excitation sequence presented in this paper. Instead of using a shaped low-intensity radiofrequency pulse, a cluster of hard 90 degrees pulses, delays of free precession, and pulsed field gradients can be used to selectively excite a narrow chemical shift range within a relatively short time. Thereby, off-resonance magnetization, which is allowed to evolve freely during the free precession intervals, is destroyed by the gradient pulses. Off-resonance excitation artifacts can be removed by random variation of the interpulse delays. This leads to an excitation profile with selectivity as well as phase and relaxation behavior superior to that of commonly used shaped-selective pulses. Since the evolution of scalar coupling is inherently suppressed during the double-selective excitation of two different scalar-coupled nuclei, the presented pulse cluster is especially suited for simultaneous highly selective excitation of N-H and C-H fragments. Experimental examples are demonstrated on hen egg white lysozyme (14 kD) and the bacterial antidote ParD (19 kD).

Magnetic Resonance Spectroscopy↗

PubMed2000

Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.

Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.

Apolipoproteins A↗

PubMed1998

Automated Processing of Two-Dimensional Correlation Spectra

An automated scheme is described which locates the centers of cross peaks in two-dimensional correlation spectra, even under conditions of severe overlap. Double-quantum-filtered correlation (DQ-COSY) spectra have been investigated, but the method is also applicable to TOCSY and NOESY spectra. The search criterion is the intrinsic symmetry (or antisymmetry) of cross-peak multiplets. An initial global search provides the preliminary information to build up a two-dimensional "chemical shift grid." All genuine cross peaks must be centered at intersections of this grid, a fact that reduces the extent of the subsequent search program enormously. The program recognizes cross peaks by examining the symmetry of signals in a test zone centered at a grid intersection. This "symmetry filter" employs a "lowest value algorithm" to discriminate against overlapping responses from adjacent multiplets. A progressive multiplet subtraction scheme provides further suppression of overlap effects. The processed two-dimensional correlation spectrum represents cross peaks as points at the chemical shift coordinates, with some indication of their relative intensities. Alternatively, the information is presented in the form of a correlation table. The authenticity of a given cross peak is judged by a set of "confidence criteria" expressed as numerical parameters. Experimental results are presented for the 400-MHz double-quantum-filtered COSY spectrum of 4-androsten-3,17-dione, a case where there is severe overlap. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Conformational behaviour of the TraM headpiece.

The structure of the headpiece of the TraM protein was investigated in different solvents. The very first 22 amino acids which alternate in their hydrophilic and hydrophobic character formed a helical structure in the presence of a membrane mimetic. In water alone the structure was flexible with a small amount of helicity according to circular dichroism measurements, whereas a loop structure was observed in dimethyl sulphoxide.

Amino Acid Sequence↗

PubMed1996

Combined use of NMR, distance geometry and MD calculations for the conformational analysis of opioid peptides of the type [D(L)-Cys2, D(L)-Cys5]enkephalin.

The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr1-Cys2-Gly3-Phe4-Cys5-OH cyclic (2-5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H-Tyr1-D-Cys2-Gly3-Phe4-D-Cys5-OH cyclic (2-5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H-Tyr1-D-Cys2-Gly3-Phe4-Cys5-OH cyclic (2-5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H-Tyr1-Cys2-Gly3-Phe4-Cys5-OH cyclic (2-5) disulfide, which contains solely L-Cys (LCLCE), there is no single structure compatible with the NMR data.

Computer Simulation↗

PubMed1985

Investigation of the hyaluronic acid-copper complex by N.M.R. spectroscopy.

Analysis of the 13C and 1H relaxation data of the hyaluronic acid-copper complex indicates a binding site involving the carboxyl group and O-1 of the D-glucuronic acid moiety. The paramagnetic relaxation of Cu2+ is discussed within the framework of the Solomon-Bloembergen formalism and it is shown that various atoms experience, in addition to the dipolar paramagnetic relaxation, a strong scalar relaxation contribution. E.s.r. spectra have also been obtained in order to determine the binding constants, and measurements at 69 K gave the g-values of the complex.

Binding Sites↗

PubMed1979

[Spin-lattice-relaxationtime T1 measurements of nucleosides (author's transl)].

Nucleosides and nucleotides have been investigated by means of the spin-lattice-relaxationtime T1, the measurements carried out in CDCl3 on the nucleosides adenosine (A), uridine (U) and inosine (I) indicate that the strength of H-bonding is highest in the basepair (A)--(U). The systems (A)--(A), (U)--(U), (I)--(A) and (I)--(U) however, show only a low tendency to associate. Plotting the spin-lattice-relaxationtime, of the nucleotide protons in fragments (mol weight below 40,000) originated from DNA or RNA, against increasing temperature gives a curve with a sigmoide shape and a Tm value of 80 degrees C. The average of the activation energy for the H-bonding is 18.02 KJ/mol. The investigations clearly show that the spin-lattice-relaxationtime measurements open first of all a new method for research on H-bonding, secondly it is even in the case of nucleosides and oligonucleotides, where most of the common methods fail, a way to get information about H-bonding effects.

Animals↗

PubMed1979

[Spin-lattice-relaxationtime-T1 measurements of hyaluronic acid (author's transl)].

Reductions with [3H5NaBH4 proof that the decrease in viscosity of hyaluronic acid solutions caused by lowering the pH does not depend on a depolymerisation of hyaluronic acid. At the same time investigations at different pH-values, show a sigmoide increase of the Spin-Lattice-Relaxationtime T1. This increase depends on a progressive aggregation of the hyaluronic acid molecule. The effect seems to be induced by the decrease of ionization of the carboxylgroups, by acidification of the solution.

Carbohydrate Conformation↗

PubMedDate not supplied

[In vitro T1- and T2-relaxation times of coagulating blood and thromboses].

A comparison between blood and thromboses is made on behalf of T1 and T2 relaxation data. Thereby, the time interval between the withdrawal and the measurement of blood forms a parameter of investigation. Among the thromboses the surgically removed ones are compared with those aggregated in blood during course of time. As the most outstanding result one can see that the formation of thromboses changes significantly the T1 and T2 relaxation times of 1H und 23Na. The values of the relaxation times obtained for those thromboses which have aggregated within the blood are thereby comparable with those relaxation times found for the surgically removed ones.

Blood Coagulation↗