A series of rigid aromatic templates that vary in size, shape, and directionality have been investigated in template-assembled synthetic 4R-helix peptide bundles for their capacity to enhance the R-helicity of an amphiphilic peptide (DAATALANALKKL-[NHCH 2 CH 2 SH]). In aqueous phosphate buffer (10 mM, pH 7) the peptide has some innate helicity (∼30%) which is concentration-independent between 1 and 250 µM. Helicity is enhanced to 64-75% when 4 equiv of the peptide are connected to aromatic templates based on benzene, benzanilide, or a cyclic octapeptide. This effect is concentration-independent by circular dichroism spectroscopy (3-60 µM [TASP]), the TASPs are monomeric by sedimentation equilibrium experiments, and have comparable thermodynamic stabilities. Thus these templates induce intra-rather than inter-molecular peptide association and are equally effective despite variations in size, shape, and directionality. When the linker between the template and peptides is sufficently long, as in these cases, TASP formation is less sensitive to the dimensions of the template than to the communication between hydrophobic peptide side chains, which are the main determinants of helix separation, 4R-helix bundle size and stability. This greatly simplifies approaches to developing small molecule mimetics of interacting protein surfaces. However template size, shape, and directionality may still be important when the linker is short or when assembled peptide surfaces are isolated from one another and unable to communicate. † Centre for Drug Design and Development.
Considerations of the effect of a small cosolute on the sedimentation equilibrium distribution for a noninteracting protein have led to the development of a simple procedure for evaluating both the molecular weight of the protein and the second virial coefficient describing the excluded volume interaction between protein and cosolute. Its application is illustrated by analysis of sedimentation equilibrium distributions for bovine thyroglobulin and horse liver alcohol dehydrogenase in the presence of a range of sucrose concentrations, and also of those for aldolase in the presence of urea to obtain the subunit molecular weight of this tetrameric enzyme. The effects of sucrose concentration on the sedimentation coefficients of thyroglobulin, catalase, and horse liver alcohol dehydrogenase are also examined to demonstrate that the second virial coefficients for protein-cosolute excluded volume interaction may be determined, albeit with less precision, from the cosolute concentration required to render the sedimentation coefficient zero by virtue of its effect on the buoyancy term. These findings serve to reinforce the fact that the effects of small cosolutes usually ascribed to changes in "protein solvation" are envisaged more realistically in terms of excluded volume.
The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron-transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4°C. Analysis of sedimentation-equilibrium distributions obtained at 15000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. P. & Winzor, D. J. (1996) Biopolymers 38, 119-1301 has yielded an intrinsic dissociation constant of 3-7 pM for the interaction of electron-transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules.Keywords: electron-transfer flavoprotein ; trimethylamine dehydrogenase ; protein interaction ; analytical ultracentrifugation.The coexistence of protein . protein complexes in dissociation equilibrium with their constitutive reactants is a general feature of interprotein-electron-transfer reactions. Whereas the specific interactions involved in the formation of tightly bound protein . protein complexes may be elucidated by NMR and Xray -crystallographic procedures, those responsible for the weaker electron-transfer complexes are poorly understood because of the inability of current structure-determination procedures to accommodate the lability of the weaker protein . protein complexes. It is thought that the components of the electron; transfer complex may associate initially in unreactive configurations via non-specific interactions, and undergo a diffusional search for the specific configuration commensurate with electron transfer, termed the speculative reduction-in-dimensionality principle. The relatively weak interaction between two physiological redox partners, trimethylamine (Me,N) dehydrogenase and its electron-transferring flavoprotein (ETF), forms an archetypal example that is an excellent model for studying interprotein electron transfer.Me,N dehydrogenase is a complex iron-sulfur flavoprotein that converts Me,N to dimethylamine and formaldehyde via the reaction [I] (CH,),N + H 2 0 -(CH,),NH + HCHO + 2e-+ 2H'.I n vivo, the electrons derived from this conversion are passed to ETF, thereby generating the oxidised form of the enzyme [2]. Both components of this redox system have been cloned and From the high-resolution structure of Me,N dehydrogenase
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