Browsing by Author "Klump, H H"

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    Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylophic yeast Pichia pastoris
    (1996) Hüsler, Jennifer; Klump, H H; Brandt, Wolf F; Maeder, Dennis
    Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Curcrbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I.
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    Circular dichroism as a means to follow DNA gymnastics:on the shoulders of giants
    (2009) Mills, M; Lin, C; Chauhan, M; Klump, H H
    This is the first report of DNA stem-loops self-assembled by 'foot-loop' interactions into either two-dimensional strings or three-dimensional spirals, distinguished by circular dichroism spectroscopy. All subunits are linked by cooperative Watson-Crick hydrogen bonds
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    DNA-mediated biomineralization of a new planar Pt-complex
    (2006) Klump, H H; Koch, K; Lin, C T
    The crystal growth morphology of a coordination complex of Pt(II) that crystallizes from solution can be controlled by using a second molecular species such as peptides or other organic compounds. Examples of crystal growth controlled by nucleic acids are few. In this article we describe the use of branched three-way junction (3WJ) DNA to influence the crystal growth of a planar platinum compound, cis-[(2, 2′-bipyridyl)N,N-di(2-hydroxyethyl)-N′-benzoylthioureatoplatinum(II)]chloride. Platinum complexes with extended planar aromatic residues are capable of stacking in the absence as well as in the presence of linear DNA double helices. This feature is based on the interaction of the compound with DNA through intercalation, resulting in the prevention of binding of DNA polymerase. Microscopic one-dimensional crystals were observed under these conditions. In the presence of the branched 3WJ DNA, however, additional nucleation sites are present, resulting in extended crystal growth of unique Pt compounds. At least two different crystal modifications were observed using transmission electron microscopy.
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    Thermodynamic characterization of DNA Triple-Helical three-way junctions
    (1995) Hüsler, Paul L; Klump, H H
    Watson-Crick CWC) and Hoogsteen (HG) triple-helical three-way junctions were constructed from three 33-mer oligonucleotides. The same sub-set of sequences have been used in the arms of the junctions. The junctions differ primary in the arrangement of the branch point and the ends of the arms. In the case of the HG triple-helical three-way junction, the three 33-mer oligonucleotides can fold into hairpin structures, linked by a four membered cytosine loop. Each of the hairpins contain a homo-pyrimidine 10-mer single strand extension which interacts with a neighboring hairpin to form a triple-helix on lowering the pH (between 6 and 4), via Hoogsteen (HG) hydrogen bonding. Collectively this process results in the formation of the branch point and the triple-helical arms. In the case of the WC triple-helical three-way junction, the three 33-mer oligonucleotides interact with a neighboring oligonucleotide to form a duplex. Collectively this process leads to the formation of a double-helical three-way junction with each arm containing a 9-mer homo-pyrimidine extension connected by four cytosines. Each of the single strand extensions can mutually fold back onto the duplex arms converting each arm into a triplex. The WC and HG triple-helical three-way junctions were characterized by gel electrophoresis, temperature gradient gel electrophoresis, circular dichroism (CD), uv melting, and differential scanning calorimetry (DSC). In both structures; arm A contained exclusively TAT triad bases, while arms B and C contained an increasing number of CGC+ triads, respectively. To counteract possible crowding at the branch point the 5' sequence was shortened by one base. The assembly of the completely folded structure was found to be spontaneous if an appropriate ionic strength and pH range was chosen. A separate set of isolated arms has been investigated to elucidate the role each arm plays in the complete structure. Comparing the summed-up properties of these arms with the data obtained for the integral three-way junctions, it is obvious that the we three-way junction is partly distorted at the branch point, in line with observation obtained from double-helical three-way junctions, while the HG threeway junction is completely ordered. A set of mathematical models has been developed to describe the thermal unfolding of the multi-strand DNA structure and to identify the intermediate states. Presented is a formalism, starting from the grand partition function, that describes the effects of pH on the thermal stability of triple-helices. The formalism can be used over a wide pH range. It covers nearest neighbor electrostatic effects of closely spaced cytosines in the Hoogsteen and Watson & Crick strands. A procedure is employed to predict enthalpy and entropy changes for triplex formation. The obtained values are in good agreement with the results obtained by differential scanning calorimetry. It is the first time that multistrand, branched DNA structures of this complexity were constructed, completely described, and characterized thermodynamically.