A designed beta-hairpin peptide for molecular recognition of ATP in water

A designed 12-residue beta-hairpin peptide with a diagonal tryptophan (Trp) pair was shown to bind ATP in water through a combination of aromatic and electrostatic interactions. The affinity for ATP was 5800 M-1 (DeltaG approximately -5.0 kcal/mol), a remarkable affinity for a short, structured peptide in water, consisting of entirely natural amino acid residues. Proton NMR measurements indicate that the adenine ring of the nucleotide is intercalated between the diagonal tryptophans in the bound state. Delineation of the contributions to ATP binding to the hairpin suggest that aromatic interactions contribute approximately -1.8 kcal/mol, while individual electrostatic interactions involving the ATP phosphates and positively charged side chains of the hairpin contribute approximately -1 kcal/mol each. The designed beta-hairpin receptor presents a novel minimalist system to investigate the energetic contributions to protein-nucleic acid recognition through the surface of a beta-sheet.     

Parameter dependence and outcome dependence in dynamical models for state vector reduction

We apply the distinction between parameter independence and outcome independence to the linear and nonlinear models of a recent nonrelativistic theory of continuous state vector reduction. We show that in the nonlinear model there is a set of realizations of the stochastic process that drives the state vector reduction for which parameter independence is violated for parallel spin components in the EPR-Bohm setup. Such a set has an appreciable probability of occurrence ( 1/2). On the other hand, the linear model exhibits only extremely small parameter dependence effects. We investigate some specific features of the models and we recall that, as has been pointed out recently, if one wants to be able to speak of definite outcomes (or equivalently of possessed objective elements of reality) at finite times, one has to slightly change the criteria for their attribution to physical systems. The concluding section is devoted to a detailed discussion of the difficulties which one meets when one tries to take, as a starting point for the formulation of a relativistic theory, a nonrelativistic scheme which exhibits parameter dependence. Here we derive a theorem which identifies the precise sense in which the occurrence of parameter dependence forbids a genuinely relativistic generalization. Finally we show how the appreciable parameter dependence of the nonlinear model gives rise to problems with relativity, while the extremely weak parameter dependence of the linear model does not give rise to any difficulty, provided one takes into account the appropriate criteria for the attribution of definite outcomes.Work supported in part by the Trieste Section of the INFN.     

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane?polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic bacterial cellulose membranes versus hydrophobic modified poly(ether)sulfone membranes. These results are discussed with reference to analysis and utilization of biofunctional membranes.     

Minimalist protein design: a beta-hairpin peptide that binds ssDNA

A 28-residue beta-hairpin dimer (WKWK)2 with two Trp and two Lys residues on one face of each beta-sheet was shown to form a complex with single-stranded oligonucleotides at low micromolar concentrations. Each beta-hairpin of the dimer contains a cross-strand Trp-Trp pair in a diagonal orientation which has previously been shown to create a cleft for the intercalation of aromatic guests such as adenine (J. Am. Chem. Soc. 2003, 125, 9580). The beta-hairpin dimer binds 5-residue ssDNA sequences 5'-AAAAA-3', 5'-TTTTT-3', and 5'-CCCCC-3' in water with dissociation constants in the range of 12-30 muM. A weak energetic preference for binding to sequence 5'-AAAAA-3' was observed, which is believed to result from stronger stacking interactions between Trp and the adenine base. The interaction of 5'-AAAAA-3' with the Lys and Trp residues of the peptide was evident by NMR, and a 1:1 association was demonstrated. The recognition of an 11-residue ssDNA sequence occurred with a dissociation constant of 3 muM under near-physiological ionic strength and pH, demonstrating that the beta-hairpin dimer binds ssDNA as strongly as many naturally occurring proteins. The salt dependence of the interaction of the 11-residue oligonucleotide with the peptide dimer indicates that Trp-nucleobase stacking interactions contribute about -4 kcal/mol to recognition, which is much greater than the contribution of nonionic interactions in unstructured peptides containing Trp. Moreover, recognition of the ssDNA demonstrated reduced salt dependence relative to the corresponding duplex, resulting in selectivity for ssDNA under high salt conditions. Peptide (WKWK)2 is a relevant mimic of OB-fold (oligonucleotide/oligosaccharide-binding) proteins which bind ssDNA on the surface of a beta-sheet.     

C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

Background  

Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.     

Adenine and thymine grafts on polyethyleneimine

Graft polymers of three different molecular weights of polyethyleneimine (PEI) with 9-(vinylsulfonylethyl) (9-VSE)-adenine and 1-(vinylsulfonylethyl) (1-VSE)-thymine were prepared. PEI's having molecular weights of 600, 1200, and 1800 were used and various ratios of VSE-nucleobases to ethyleneimine were employed in the grafting process. As expected, a decrease in the nucleobase substitution in the resulting graft polymer resulted in an increase in its water solubility. The degree of substitution in the graft polymers was calculated from sulfur determinations: in most cases this degree of substitution indicated that not all of the VSE derivative has reacted with the PEI due to the highly branched nature of the latter. The presence of nucleobase stacking in the polymers was studied by UV spectroscopy. Stacking was observed for the adenine polymers in both DMSO and 0.1N HCl while stacking in the thymine polymers was observed in 0.1N HCl but not in DMSO. The absence of stackingwithin the thymine polymers in DMSO was attributed to complexation of primary amino groups of the PEI backbone with the pyrimidine ring.