The major coat protein (gVIIIp) of bacteriophage M13 solubilized in sodium dodecyl sulfate (SDS) detergent micelles was used as a model system to study this protein in the lipid-bound form. In order to probe the position of gVIIIp relative to the SDS micelles, stearate was added, spin-labeled at the 5- or 16-position with a doxyl group containing a stable nitroxide radical. The average position of the spin-labels in the micelles was derived from the line broadening of the resonances in the 13C spectrum of SDS. Subsequently, we derived a model of the relative position of gVIIIp in the SDS micelle from the effect of the spin-labels on the gVIIIp resonances, monitored via 1H-15N HSQC and TOCSY experiments. The results are consistent with the structure of gVIIIp having two helical strands. One strand is a long hydrophobic helix that spans the micelle, and the other is a shorter amphipathic helix on the surface of the micelle. These results are in good agreement with the structure of gVIIIp in membranes proposed by McDonnell et al. on the basis of solid state NMR data [McDonnell, P. A., Shon, K., Kim, Y., & Opella, S. J. (1993) J. Mol. Biol. 233, 447-463]. This study indicates that high-resolution NMR on this membrane protein, solubilized in detergent micelles, is a very suitable technique for mimicking these proteins in their natural environment. Furthermore, the data indicate that the structure of the micelle near the C-terminus of the major coat protein is distorted.(ABSTRACT TRUNCATED AT 250 WORDS)

In this paper, a detailed description is presented of the aromatic part of the 500-MHZ 1H nuclear magnetic resonance (NMR) spectrum of the helix-destabilizing gene-5 protein (GVP) encoded by the coliphage M13. As a result of the resolution obtained at 500 MHZ, it was possible to perform selective decoupling and time-resolved selective Overhauser experiments. The magnitudes of the observed Overhauser effects compare favorably with magnitudes expected on the basis of theoretical calculations. These experiments in conjunction with selective decoupling experiments allowed a detailed interpretation of the aromatic part of the protein spectrum. The spectrum of the aromatic part of the GVP-d(A)8 complex could be interpreted in a similar fashion. The ring protons of one phenylalanyl residue and of two tyrosyl residues show rather large shifts upon complex formation. This indicates that these residues are involved in the interaction with the DNA molecule in accordance with earlier observations. Direct evidence for the proximity of these aromatic rings and the DNA fragment in the complex was obtained by additional Overhauser experiments. It turns out that the H3',H4', and/or the H5' sugar protons of the oligonucleotide are situated near the ring protons of (most likely) two or all three of the aromatic residues of which the resonances undergo large shifts upon complex formation.

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

SummaryA new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H 8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance 13C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular confor mations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.

The binding of the bacteriophage-M13-encoded gene-5 protein to oligo(deoxythymidy1ic acid)s and MI 3 DNA was studied by means of tyrosyl fluorescence decay and fluorescence anisotropy measurements. The observed fluorescence decays could be described with two exponentials, characterised by the lifetimes T~ = 2.2 ns and z2 = 0.8 ns respectively. Only the amplitude of the longer-lifetime component is influenced by binding of the protein to DNA. This indicates that a part of the tyrosyl residues is involved in the binding. By means of fluorescence depolarisation measurements the rotational correlation time of the protein dimer is found to be 12.9 ns. In contrast to earlier measurements, carried out on the DNA- [85][86][87][88][89][90][91][92][93], the observed rotational correlation times of the gene-5 protein pass through a maximum when the protein is titrated with oligo(deoxythymidy1ic acid)s. This is not observed upon titration with M13 DNA. Our measurements showed that for the oligo(deoxythymidylic acid)s there clearly is a decrease in the number of clustered proteins on the lattice in the case of excess nucleotide. This is a direct consequence of the much lower cooperativity of the binding to the oligonucleotides compared to the cooperativity characteristic of binding to polynucleotides. The number of nucleotides covered by a protein monomer is found to be I 3 for the oligonucleotides and % 4 for M13 DNA. Model calculations show that the 'time-window' through which the fluorescence depolarisation can be observed (i.e. the fluorescence lifetime) in this case significantly affects the 'measured' effective rotational correlation times.An interesting aspect of the interaction between the singlestrand-DNA-binding gene-5 protein, encoded by the bacteriophage M13, is its ability to bind to DNA in two different modes, previously designated the oligonucleotide and the polynucleotide binding mode. In the polynucleotide binding mode the protein covers four nucleotides and the cooperativity of binding is high, i.e. the binding of a protein adjacent to an already bound protein is increased by a factor of five hundred (o = 500) with respect to binding to a naked lattice. In the so-called oligonucleotide binding mode the protein covers three nucleotides and the cooperativity of the binding is two orders of magnitude smaller (w w 5). Moreover, the salt dependence of the two binding modes differs significantly [l-31. Recently the binding characteristics of the single-strand-DNA-binding protein of the filamentous phage Pfl were studied in detail by means of fluorescence depolarisation measure- DNA. However, in contrast to the M13 gene-5 protein, for the Pfl protein different binding modes could not be distinghuished: complex formation to polynucleotides and oligonucleotides turned out to be very much the same. Thus the question arises whether the two proteins are intrinsically different in their DNA-binding properties. This is not self evident, because the second binding mode discovered for the M13 gene-5 protein was detected...