A two-step strategy to enhance activity of low potency peptides.

Novel strategies are needed to expedite the generation and optimization of peptide probes targeting G protein-coupled receptors (GPCRs). We have previously shown that membrane tethered ligands (MTLs), recombinant proteins comprised of a membrane anchor, an extracellular linker, and a peptide ligand can be used to identify targeted receptor modulators. Although MTLs provide a useful tool to identify and/or modify functionally active peptides, a major limitation of this strategy is the reliance on recombinant protein expression.

Fluorination in the design of membrane protein assemblies.

Protein design approaches based on the binary patterning of nonpolar and polar amino acids have been successful in generating native-like protein structures of amphiphilic α-helices or idealized amphiphilic β-strands in aqueous solution. Such patterning is not possible in the nonpolar environment of biological membranes, precluding the application of conventional approaches to the design of membrane proteins that assemble into discrete aggregates. This review surveys a promising, new strategy for membrane protein design that exploits the unique properties of fluorocarbons-in particular, their ability to phase separate from both water (due to their hydrophobicity) and hydrocarbons (due to their lipophobicity)-to generate membrane protein assemblies.

Ligand-induced protein mobility in complexes of carbonic anhydrase II and benzenesulfonamides with oligoglycine chains.

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1-5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with (15)N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly.

A non-chromatographic method for the purification of a bivalently active monoclonal IgG antibody from biological fluids.

This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgG(DNP); and mouse antidigoxin IgG: IgG(Dgn)) from ascites fluid. This procedure (for IgG(DNP)) has three steps: (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation of cyclic complexes of IgG(DNP) by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; (iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid.

Thermodynamic parameters for the association of fluorinated benzenesulfonamides with bovine carbonic anhydrase II.

This paper describes a calorimetric study of the association of a series of seven fluorinated benzenesulfonamide ligands (C6H(n)F(5-n)SO2NH2) with bovine carbonic anhydrase II (BCA). Quantitative structure-activity relationships between the free energy, enthalpy, and entropy of binding and pKa and log P of the ligands allowed the evaluation of the thermodynamic parameters in terms of the two independent effects of fluorination on the ligand: its electrostatic potential and its hydrophobicity. The parameters were partitioned to the three different structural interactions between the ligand and BCA: the Zn(II) cofactor-sulfonamide bond (approximately 65% of the free energy of binding), the hydrogen bonds between the ligand and BCA (approximately 10%), and the contacts between the phenyl ring of the ligand and BCA (approximately 25%).

Dependence of effective molarity on linker length for an intramolecular protein-ligand system.

This paper reports dissociation constants and "effective molarities" (M(eff)) for the intramolecular binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EG(n)) linkers of different lengths (n = 0, 2, 5, 10, and 20) and compares these experimental values with theoretical estimates from polymer theory. As expected, the value of M(eff) is lowest when the linker is too short (n = 0) to allow the ligand to bind noncovalently at the active site of the protein without strain, is highest when the linker is the optimal length (n = 2) to allow such binding to occur, and decreases monotonically as the length increases past this optimal value (but only by a factor of approximately 8 from n = 2 to n = 20). These experimental results are not compatible with a model in which the single bonds of the linker are completely restricted when the ligand has bound noncovalently to the active site of the protein, but they are quantitatively compatible with a model that treats the linker as a random-coil polymer.

Designing ligands to bind proteins.

The ability to design drugs (so-called 'rational drug design') has been one of the long-term objectives of chemistry for 50 years. It is an exceptionally difficult problem, and many of its parts lie outside the expertise of chemistry. The much more limited problem - how to design tight-binding ligands (rational ligand design) - would seem to be one that chemistry could solve, but has also proved remarkably recalcitrant.

The paradoxical thermodynamic basis for the interaction of ethylene glycol, glycine, and sarcosine chains with bovine carbonic anhydrase II: an unexpected manifestation of enthalpy/entropy compensation.

This paper describes a systematic study of the thermodynamics of association of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamides with chains of oligoglycine, oligosarcosine, and oligoethylene glycol of lengths of one to five residues. For all three of these series of ligands, the enthalpy of binding became less favorable, and the entropy less unfavorable, as the chain length of the ligands increased. The dependence on chain length of the enthalpy was almost perfectly compensated by that of the entropy; this compensation resulted in dissociation constants that were independent of chain length for the three series of ligands.

Promotion of opsonization by antibodies and phagocytosis of Gram-positive bacteria by a bifunctional polyacrylamide.

This paper describes the application of a bifunctional polyacrylamide (pA-V-F) presenting both vancomycin and fluorescein groups, to modify the surfaces of multiple species of Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis) to control molecular recognition of these surfaces. The vancomycin groups allowed the specific recognition of a structural component of the bacterial cell wall: peptides terminated in D-Ala-D-Ala. The fluorescein groups allowed the imaging of binding of polymer to the surfaces of bacteria by fluorescence, and are representative, low molecular weight haptens; their recognition by anti-fluorescein antibodies provides proof-of-principle that bifunctional polymers can be used to introduce haptens onto the surface of the bacteria.

Synthesis of monodisperse polymers from proteins.

Proteins are functional biopolymers; viewed as molecules, they are also monodisperse polyamides with chemically reactive side chains. This paper describes the use of proteins as starting materials for the synthesis of monodisperse polymers with nonbiological functionalities attached to the side chains. It demonstrates the complete derivatization of amine groups (lysine side chains and N-termini) on three different proteins by addition of activated carboxylate reagents in aqueous solutions containing sodium dedecyl sulfate (SDS), under denaturing conditions.