William H. Braunlin

SPECTROSCOPIC STUDIES OF A PHOSPHOINOSITIDE-BINDING PEPTIDE FROM GELSOLIN : BEHAVIOR IN SOLUTIONS OF MIXED SOLVENT AND ANIONIC MICELLES

The peptide G(150-169) corresponds to a phosphatidylinositol 4,5-bisphosphate (PIP2) and filamentous actin (F-actin) binding site on gelsolin (residues 150-169, with the sequence KHVVPNEVVVQRLFQVKGRR). The conformation of this peptide in trifluoroethanol (TFE) aqueous solution was determined by 1H nuclear magnetic resonance as the first step toward understanding the structural aspects of the interaction of G(150-169) and PIP2. The circular dichroism experiments show that G(150-169) adopts a predominantly alpha-helical form in both 50% TFE aqueous solution and in the presence of PIP2 micelles, therefore establishing a connection between the two conformations. 1H nuclear magnetic resonance experiments of G(150-169) in TFE co-solvent show that the helical region extends from Pro-154 to Lys-166. The amphiphilic nature of this helical structure may be the key to understanding the binding of the peptide to lipids. Sodium dodecyl sulfate micelle solution is used as a model for anionic lipid environments. Preliminary studies of the conformation of G(150-169) in sodium dodecyl sulfate micelle solution show that the peptide forms an alpha-helix similar to but with some structural differences from that in TFE co-solvent. Fluorescence experiments provide evidence of peptide clustering over a narrow range of peptide/PIP2 ratios, which is potentially relevant to the biological function of PIP2.

Ca2+ binding environments on natural and synthetic polymeric DNA's

Here are reported 43Ca nmr chemical shift and line width measurements obtained during 43CaClO4 titrations of two natural and two synthetic polymeric DNAs. Titrations of the natural DNAs demonstrate the existence of at least two classes of bound 43Ca2+. The 43Ca2+ nmr relaxation and chemical shift behavior observed during titration of C. perfringens DNA (31%GC) is dominated by a delocalized, non-specific interaction. In contrast, titration of M. lysodeikticus DNA (72% GC) indicates that a small fraction of the 43Ca2+ experiences significant motional retardation and/or an increase in the electric field gradient when associated to the DNA, and thus appears to be locally bound to discrete sites on the DNA. These results, and previous results for calf thymus DNA (39% GC) demonstrate that higher GC content correlates with an increase in favorable Ca2+ binding environments. Titrations of synthetic DNA demonstrate that Ca2+ binding is remarkably sensitive to local DNA structure.

Trifluoperazine binding to calmodulin: A shift reagent 43Ca NMR study

43Ca NMR experiments of Ca2+ binding to calmodulin (CaM) were performed in the presence and absence of the calmodulin antagonist trifluoperazine (TFP). By making use of the shift reagent Dy(PPP)(7-) (a 1:2 complex of DyCl3 and Na5P3O10) we have succeeded in separating the 43Ca resonances of protein-bound Ca2+ and free Ca2+ in the otherwise unresolved spectra. This experimental strategy has allowed us to demonstrate unequivocally that the affinity of CaM for Ca2+ is markedly increased in the presence of TFP. Thus Ca2+ is not liberated from the protein upon addition of TFP as had been suggested based on earlier 43Ca NMR experiments (Shimuzu, T., Hatano, M., Nagao, S. and Nozawa, Y. (1982), Biochem. Biophys. Res. Comm. 106, 1112-1118).

Metal Ion NMR: Application to Biological Systems

Publisher Summary This chapter discusses the biological importance of metal ion nuclear magnetic resonance (NMR). Inorganic elements have a multitude of functions in biological systems, such as carriers of ionic signals, triggering of proteins, formation of structures, electron transport, and enzyme catalysis. All essential elements in biological systems and all elements of the periodic table have at least one potentially valuable isotope for NMR studies. This is because the NMR sensitivity of many biologically interesting nuclei is very low. NMR parameters are generally dependent upon the chemical nature of the nuclear environment and on its structure and dynamics. Thus, NMR spectroscopy may be used to identify and characterize metal ion binding sites on biological macromolecules and how these change during the course of biochemical reactions. The possibility of using NMR to determine metal ion populations of individual binding sites on a multisite protein is not matched by many other physical techniques. Metal ion NMR also makes it possible to address such elusive questions as cooperatively or anticooperativity of ion binding. As the relaxation of quadrupolar nuclei usually is caused by fluctuating electrical field gradients at the site of the nucleus, NMR studies can provide insight into ion binding to poly electrolytes, discriminate between nonspecific and specific ion binding, and are in general useful for testing theoretical models of ion binding

Shift reagents for calcium-43 NMR studies of calcium-binding proteins

Abstract Ca 2+ ions binding to calcium-binding proteins usually experience a small chemical shift and a large broadening in 43 Ca NMR spectra. In order to minimize problems arising from overlapping resonances in such spectra, enhancement of the resolution by shifting resonances for free or weakly bound rapidly exchanging Ca 2+ ions has been attempted. Upfield shifts of up to −125 ppm and downfield shifts of up to 60 ppm were obtained upon titration of Ca 2+ standard solutions with various lanthanide-chelator complexes. Most effective were the Dy 3+ and Tm 3+ complexes with tripolyphosphate. The induced hyperfine shifts are shown to be mainly of dipolar origin. High salt concentrations or pH values lower than 7 reduced the effectiveness of the shift reagents. When these compounds were used in samples containing the calcium-binding proteins, bovine a-lactalbumin and parvalbumin, the introduction of the shift reagent dramatically improved the spectral resolution: resonances for rapidly exchanging weakly bound Cap 2+ ions experienced shifts, while those for the tightly bound slowly exchanging Ca 2+ ions were not affected. It is suggested that the same strategy for improving spectral resolution could be useful in studies with other quadrupolar nuclei.

AN NMR SELF-DIFFUSION STUDY OF THE INTERACTIONS BETWEEN SPERMIDINE AND OLIGONUCLEOTIDES

Self‐diffusion coefficients have been determined by pulsed field gradient nmr methods for spermidine in solutions of the oligonucleotides d(GC)4 and d(GGAATTCC). The self‐diffusion behavior of spermidine in solution of d(GC)4 is very similar to that observed previously for methylspermidine (completely N‐methylated spermidine). Moreover, the self‐diffusion behaviors of spermidine in solutions of d(GC)4 and d(GGAATTCC) are also quite similar, indicating that there is no significant influenceon on self‐diffusion of oligonucleotide base composition. Furthermore, self‐diffusion coefficients of the oligonucleotide d(GC)8 show only a small dependence on oligonucleotide concentration, and no measurable dependence on sodium ion or magnesium ion concentration.