Ben J. M. Harmsen

Structure of the DNA binding wing of the gene-V encoded single-stranded DNA binding protein of the filamentous bacteriophage M13

The structure in solution of a β‐loop in mutant Y41H of the single‐stranded DNA binding protein encoded by gene‐V of the filamentous phage M13 has been elucidated using 2‐dimensional 1H‐nuclear magnetic resonance techniques. Furthermore, these studies enabled us to demonstrate that an identical structural element is present in wild‐type gene‐V‐protein and that this element intimately is involved in the binding of gene‐V‐protein to single‐stranded DNA. It is shown that the structure of the DNA binding wing deviates from that proposed for the same amino acid sequence on the basis of X‐ray diffraction data. The structure is, however, identical to that of the DNA binding wing present in the single‐stranded DNA binding protein encoded by the genome of the evolutionary distantly related filamentous phage IKe. The latter observations support our current view that in the binding of these proteins to single‐stranded DNA a common structural motif is involved.

DNA-binding properties of gene-5 protein encoded by bacteriophage M13. 2. Further characterization of the different binding modes for poly- and oligodeoxynucleic acids.

The binding of gene-5 protein, encoded by bacteriophage M13, to oligodeoxynucleic acids was studied by means of fluorescence binding experiments, fluorescence depolarization measurements and irreversible dissociation kinetics of the protein.nucleotide complexes with salt. The binding properties thus obtained are compared with those of the binding to polynucleotides, especially at very low salt concentration. It appears that the binding to oligonucleotides is always characterized by a stoichiometry (n) of 2-3 nucleotides/protein, and the absence of cooperativity. In contrast the protein can bind to polynucleotides in two different modes, one with a stoichiometry of n = 3 in the absence of salt and another with n = 4 at moderate salt concentrations. Both modes have a high intramode cooperativity (omega about 500) but are non-interacting and mutually exclusive. For deoxynucleic acids with a chain length of 25-30 residues a transition from oligonucleotide to polynucleotide binding is observed at increasing nucleotide/protein ratio in the solution. The n = 3 polynucleotide binding is very sensitive to the ionic strength and is only detectable at very low salt concentrations. The ionic strength dependency per nucleotide of the n = 4 binding is much less and is comparable with the salt dependency of the oligonucleotide binding. Furthermore it appears that the influence of the salt concentration on the oligonucleotide binding constant is to about the same degree determined by the effect of salt on the association and dissociation rate constants. Model calculations indicate that the fluorescence depolarization titration curves can only be explained by a model for oligonucleotide binding in which a protein dimer binds with its two dimer halves to the same strand. In addition it is only possible to explain the observed effect of the chain length of the oligonucleotide on both the apparent binding constant and the dissociation rate by assuming the existence of interactions between protein dimers bound to different strands. This results in the formation of a complex consisting of two nucleotide strands with protein in between and stabilized by the dimer-dimer interactions.

500 MHz 1H NMR study of the role of lysines and arginines in the binding of gene-5 protein to oligoadenylic acids

Gene-5 protein encoded by the bacteriophages M13 fd and f~ is a DNA-hetLx destabilizing protein. It plays an essential role in the replication process of the phage, where the binding of gene-5 protein to the viral DNA strand is required for single-stranded viral DNA synthesis [1-3 ]. The dimeric protein [4,5 ] with subunit M r 9690 [6,7] binds cooperatively [1,5] to single stranded DNA thereby covering ~4 nucleotides/ protein subunit [1,4,5,8]. There is a wealth of spectroscopic evidence that aromatic residues are involved in the interaction with DNA [4,8,9]. The molecular surroundings of 2 tyrosines and 1 phenylalanine are influenced by binding of DNA [10,11]. This phenylalanine and i>1 of these tyrosines are located in close proximity to the DNA [10]. However, the interaction of gene-5 protein with DNA is strongly dependent on ionic strength [1]. One therefore expects that electrostatic interactions are also important in the binding. The most likely candidates for the interaction with the negatively-charged phosphate groups of the DNA are lysines and arginines. To investigate the influence of DNA binding on these residues we have performed 1H NMR experiments at 500 MHz with gene-5 protein and its complex with oligoadenylic acids.

Relationship between the conformation of bovine serum albumin and its digestion by pepsin

Abstract 1. 1.The digestion of bovine serum albumin by pepsin in the pH region of the Normal-Fast transition was studied by means of electrophoresis on polyacrylamide gels. The results suggest a close relationship between the conformation of the albumin molecule and its digestion by pepsin. 2. 2.At pH values just above 3.6 ten well-defined fragments were found. These features might support a model of albumin in which there are only a few, possibly three sites, available for hydrolysis by pepsin. 3. 3.At pH values below 3.6 the more complex electrophoretic patterns suggest the existence of more sites, probably as a consequence of unfolding of the albumin molecule.