Protein engineering

About: Protein engineering is a research topic. Over the lifetime, 4558 publications have been published within this topic receiving 232470 citations.

Introduction to protein structure

Abstract: PART 1 BASIC STRUCTURAL PRINCIPLES 1. The Building Blocks 2. Motifs of Protein Structure 3. alpha-Domain Structures 4. alpha/ss Structures 5. ss Structures 6. Folding and Flexibility 7. DNA Structures PART 2 STRUCTURE, FUNCTION AND ENGINEERING 8. DNA Recognition in Procaryotes by Helix-Turn-Helix Motifs 9. DNA Recognition by Eukaryotic Transcription Factors 10. Specific Transcription Factors Belong to a Few Families 11. An Example of Enzyme Catalysis: Serine Proteinases 12. Membrane Proteins 13. Signal Transduction 14. Fibrous Proteins 15. Recognition of Foreign Molecules by the Immune System 16. The Structure of Spherical Viruses 17. Prediction, Engineering, and Design of Protein Structures 18. Determination of Protein Structures

Using circular dichroism spectra to estimate protein secondary structure

Abstract: Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing < or =20 microg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.

Structure and Mechanism in Protein Science

Abstract: The three-dimensional structure of proteins chemical catalysis the basic equations of enzyme kinetics measurement and magnitude of enzymatic rate constants the pH dependence of enzyme catalysis practical kinetics detection of intermediaries in reactions by kinetics stereochemistry of enzymic reactions active-site-directed and enzyme-activated irreversible inhibitors - affinity labels and suicide inhibitors conformational change, allosteric regulation, motors and work forces between molecules, and enzyme-substrate binding energies enzyme-substrate complementarity and the use of binding energy in catalysis specificity and editing mechanisms recombinant DNA technology case studies of enzyme structure and mechanism protein engineering protein stability kinetics of protein folding folding pathways and energy landscapes.

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

Abstract: A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Abstract: Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.