In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Directed evolution of novel binding proteins

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

Methods for producing members of specific binding pairs

Self-assembled monolayers (SAMs) of ω-functionalized long-chain alkanethiolates on gold films are excellent model systems with which to study the interactions of proteins with organic surfaces. Mon...

https://science.sciencemag.org/content/sci/252/5009/1164.full.pdf

Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces

Molecular recognition is central to all biological processes. For the past 50 years, Koshland's 'induced fit' hypothesis has been the textbook explanation for molecular recognition events. However, recent experimental evidence supports an alternative mechanism. 'Conformational selection' postulates that all protein conformations pre-exist, and the ligand selects the most favored conformation. Following binding the ensemble undergoes a population shift, redistributing the conformational states. Both conformational selection and induced fit appear to play roles. Following binding by a primary conformational selection event, optimization of side chain and backbone interactions is likely to proceed by an induced fit mechanism. Conformational selection has been observed for protein-ligand, protein-protein, protein-DNA, protein-RNA and RNA-ligand interactions. These data support a new molecular recognition paradigm for processes as diverse as signaling, catalysis, gene regulation and protein aggregation in disease, which has the potential to significantly impact our views and strategies in drug design, biomolecular engineering and molecular evolution.

/pdf/the-role-of-dynamic-conformational-ensembles-in-biomolecular-33rwd16x5b.pdf

The role of dynamic conformational ensembles in biomolecular recognition

: The unpolarized absorption and circular dichroism spectra of the fundamental vibrational transitions of the chiral molecule, 4-methyl-2-oxetanone, are calculated ab initio. Harmonic force fields are obtained using Density Functional Theory (DFT), MP2, and SCF methodologies and a 5S4P2D/3S2P (TZ2P) basis set. DFT calculations use the Local Spin Density Approximation (LSDA), BLYP, and Becke3LYP (B3LYP) density functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with experiment. The MP2 force field yields spectra in slightly worse agreement with experiment than the B3LYP force field. The SCF force field yields spectra in poor agreement with experiment.The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreements with experiment. jg

Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields

A revised mechanism for the alkaline phosphatase reaction involving three metal ions.

http://www.chem.uwec.edu/Chem453_F05/453-2006/Day%208/APpaper.pdf

A model of the transition state in the alkaline phosphatase reaction.

The x-ray structures of the allosteric enzyme aspartate transcarbamylase from Escherichia coli have been solved and refined for both allosteric forms. The T form was determined in the presence of the heterotropic inhibitor cytidine triphosphate, CTP, while the R form was determined in the presence of the bisubstrate analog N-phosphonacetyl-L-aspartate. These two x-ray structures provide the starting point for an understanding of how allosteric enzymes are able to control the rates of metabolic pathways. Insights into the mechanisms of both catalysis and homotropic cooperativity have been obtained by using site-directed mutagenesis to probe residues thought to be critical to the function of the enzyme based on these x-ray structures.

https://science.sciencemag.org/content/sci/241/4866/669.full.pdf

Escherichia coli aspartate transcarbamylase: the relation between structure and function

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2899%2901448-9

Evan R. Kantrowitz

Papers

A revised mechanism for the alkaline phosphatase reaction involving three metal ions.

A model of the transition state in the alkaline phosphatase reaction.

Escherichia coli aspartate transcarbamylase: the relation between structure and function

The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis

Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition