Per-Olof Lycksell

Demonstration of segmental mobility in the functionally essential car☐yl terminal part of ribonucleotide reductase protein R2 from Escherichia coli

The C‐terminus of protein R2 is important for the formation of the enzymatically active complex between proteins R1 and R2 of ribonucleotide reductase from Escherichia coli. Some residues in this part of R2 may also be involved in intramolecular electron transfer. We now demonstrate that 26 amino acid residues at C‐terminus of protein R2 are mobile in the free protein, and can be studied by1H NMR. Spectral assignment of narrow resonances was made by comparison of TOCSY and NOESY spectra from wild‐type R2 with corresponding spectra of a mutant protein R2, lacking 30 residues at the car☐yl terminus.

SOLVENT STABILIZED SOLUTION STRUCTURES OF GALANIN AND GALANIN ANALOGS, STUDIED BY CIRCULAR DICHROISM SPECTROSCOPY

Circular dichroism spectroscopy has been used to study how different solvents stabilize secondary structure in the neuropeptide galanin (rat), two N-terminal fragments of galanin, galanin(1-12) and galanin(1-16), and six other differently charged analogs. Among these analogs, the peptide M40, galanin(1-13)-Pro-Pro-Ala-Leu-Ala-Leu-Ala amide, is a high affinity, receptor subtype specific galanin receptor antagonist. The different solvents include sodium dodecyl sulfate (SDS) micelle solutions, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) vesicle solutions. 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and 100% 2,2,2-trifluoroethanol (TFE). DOPC vesicles did not change the structure of the peptides as compared to aqueous solvent. The negatively charged DOPG vesicles and SDS micelles induced similar changes towards alpha-helical structures in all peptides. The HFP and TFE solvents have an even stronger tendency to stabilize alpha-helical conformations in these peptides. Since DOPG vesicles can be considered as a model system for negatively charged biological membranes, the solution structures observed in the presence of DOPG or SDS may be the most relevant for the in vivo situation. Correlations between the binding affinity of the peptides to hippocampal galanin receptors and their observed structures in the DOPG solvent were investigated.

A refined three-dimensional solution structure of a carboxy terminal fragment of apolipoprotein CII.

The three-dimensional structure of a synthetic fragment of human apolipoprotein CII (apo-CII) in 35%, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) has been determined on the basis of distance and intensity constraints derived from two-dimensional proton nuclear magnetic resonance measurements. The NOE crosspeak build-up rates were converted to distance constraints which were used in the distance geometry program DIANA. A set of one hundred structures were generated and of these ten structures were used in molecular dynamics simulations using the program XPLOR. This program enabled a direct minimization between the difference of the two-dimensional NOE intensities and those calculated from the full relaxation matrix. In this way spin diffusion is fully taken into account, which can be seen from the considerable improvement of the R-factor after the relaxation matrix refinement. These calculations show that this fragment, which corresponds to the carboxy terminal 30 amino acids of intact apo-CII and which retains its ability to activate lipoprotein lipase, is essentially flexible, but has three defined secondary structural elements. The most significant one is an α-helix between residues 67 and 74. The following three residues adopt a turn-like structure. Another turn of α-helix is seen between residues 56 and 59. The effect of the solvent system on the secondary structure was studied by circular dichroism spectroscopy. The results show that the mixed aqueous 35% HFP solvent induces secondary structure of a very similar nature to the one induced by sodium dodecyl sulphate.

Observation of excimer formation in the covalent adducts of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol with poly(dG-dC)

The covalent binding of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol to alternating poly(dG–dC) results in extensive occurrence of excimers as shown by fluorescence spectroscopy; the origin is inferred to be close-lying pyrene chromophores bound to guanines in the minor groove of DNA.

Structure of Z-DNA in solution. A flow linear dichroism study

U.v. flow linear dichroism (L.D.) of the high-salt form of duplex poly(dG-dC)(‘Z-DNA’) shows that the reduced dichroism of Z-DNA is about twice as large as for B-DNA and the spectrum has the shape expected for Watson–Crick base-pairs oriented perpendicular to the helix; axis spectral features observed during the B→Z conversion are in qualitative agreement with a mechanism in which the base-pairs flip 180° around the glycosyl bonds.

1H, 13C and 15N Assignment of the Isl-1 Homeodomain

Key words: Isl-1, homeodomain, protein, DNA-binding, NMR assignmentBiological contextTheinsulin geneenhancerbindingprotein, Isl-1, bindsspecifically to a transcription activation domain of theinsulin gene in rat (Karlsson et al., 1990), acts asa positive regulator of the proglucagon genes (Wangand Drucker, 1995), and is required for embryonicneuron differentiation (Pfaff et al., 1996). Isl-1 has amolecular weight of 42 kDa, and contains two aminoterminal zinc-binding LIM domains (Karlsson et al.,1990; McGurie et al., 1992), and a C-terminal regionrelated to homeodomains in other DNA binding pro-teins. The homeodomain region of Isl-1, Isl-1-HD (66amino acids), mediates its DNA binding through se-quencespecific recognitionof duplexDNA containinga –TAAT– sequence (Behravan et al., 1997). We havenow used NMR to determine the solution structure ofthe Isl-1-HD (Ippel et al., to be published), while stud-ies on the Isl-1-HD/DNA complexare still in progress.Here we report the assignment of the

The B - Z Transition in Poly [d(G-C) d(G-C)] After Covalent Binding of Anti- Benzo(a)Pyrenediolepoxide

(+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) is a highly carcinogenic metabolite of benzo(a)pyrene (BP), which binds covalently and with high specificity to the exocyclic amino group of guanine in DNA (1) (Fig. 1).