Section B: Protein StructureYang Xu, College of Life Sciences Section B Protein Structure B2 Protein structure B3 Protein Analysis

Section B: Protein StructureYang Xu, College of Life Sciences B2 Protein structure Primary structure Secondary structure Tertiary structure Quaternary structure Domains and motifs

Section B: Protein StructureYang Xu, College of Life Sciences Primary structure Definition: –the sequence of amino acids from the N to the C terminus is the primary structure of the polypeptide. Amide bond (peptide bond ) Dipeptide Polypeptide Sizes for single polypeptide chains: –within the range amino acids, though longer and shorter ones exist.

Section B: Protein StructureYang Xu, College of Life Sciences Secondary structure Definition: Since the formation of the hydrogen bonds (C=ON-H) between peptide bond units of the polypeptide backbone, polypeptides fold into several regular structures, include:  -helix: The polypeptide backbone forms a right-handed helix with 3.6 amino acid residues per turn so that each peptide N-H group is hydrogen bonded to the C=O group of the peptide bond three residues away

Section B: Protein StructureYang Xu, College of Life Sciences Secondary structure  -pleated sheet (  -sheet): is formed by hydrogen bonding of the peptide bond N － H and C=O groups to the complementary groups of another section of the polypeptide chain, include parallel  -Sheets and antiparallel  -Sheets.

Section B: Protein StructureYang Xu, College of Life Sciences Tertiary structure Definition: The different sections of  -helix,  - sheet, and other minor secondary structures and connecting loops fold in three dimensions, it is the tertiary structure of the polypeptide. The bond forces: Various types of non-covalent forces between side chains hold the tertiary structure together: van der Waals forces, hydrogen bonds, salt bridges.

Section B: Protein StructureYang Xu, College of Life Sciences Tertiary structure In addition, covalent di-sulfide bonds can form between two cysteine residues which may be far apart in the primary structure but close together in the folded tertiary structure.

Section B: Protein StructureYang Xu, College of Life Sciences Quaternary structure Two or more polypeptides

Section B: Protein StructureYang Xu, College of Life Sciences Motifs and domains Motifs Motifs are groupings of secondary structure. Similar structural motifs are also found in proteins which have no sequence similarity. Domains Domains form semi-independent structural and functional units within a single polypeptide chain.

Section B: Protein StructureYang Xu, College of Life Sciences B3 Protein Analysis Protein purification –Size –Charge –Affinity –Two dimensional electrophoresis Protein sequencing

Section B: Protein StructureYang Xu, College of Life Sciences Size Ultra-centrifugation It can be used to separate proteins but is also a powerful analytical tool for studying protein structure. Gel filtration chromatography The columns filled with a suspension of beads containing particular size pores － a molecular sieve. –Protein molecules larger than the pores elute at the bottom relatively quickly. –Protein molecules smaller than the pore can enter the beads and so have a larger volume of buffer through which they can diffuse and out. Thus, smaller proteins elute from the column after larger ones.

Section B: Protein StructureYang Xu, College of Life Sciences C harge Electrophoresis: protein mixture is applied to a gel and an electric field across the gel. Depending on their net charge, proteins will travel at different rates towards the anode or cathode and can be recovered from the gel after separation. Ion-exchange chromatography: ions that are electrostatically bound to the ion exchanger packed in a column are reversibly replaced with charged proteins from solution. Then, a salt gradient of increasing ionic strength is passed through the column and the bound proteins elute separately. protein DNA & RNA

Section B: Protein StructureYang Xu, College of Life Sciences Affinity High specificities: –enzyme-substrate –receptor-ligand –antibody-antigen For example: –a column made of a support to which the hormone insulin is covalently linked will specifically bind the insulin receptor and no other protein.

Section B: Protein StructureYang Xu, College of Life Sciences First dimensional electrohporesis Second dimensional electrohporesis Spots picker Two dimensional electrophoresis

Section B: Protein StructureYang Xu, College of Life Sciences Principle - + PI - + WM First dimensional electrophoresis (IPG) Second dimensional electrophoresis (SDS-PAGE) SDS-PAGE ： Sodium dodecyl sulfate (SDS) Polyacrylamide gel electrophoresis (PAGE)

Section B: Protein StructureYang Xu, College of Life Sciences The experiments of 2-DE

Section B: Protein StructureYang Xu, College of Life Sciences Protein sequencing Protein sequencing : –Applying a protein sequencer: Each peptide is then subjected to sequential Edman degradation in a protein sequencer. cDNA sequencing : –mRNA  cDNA  protein sequence –This is simpler and faster but misses post-translational modifications Frederick Sanger received two Nobel prizes for his work on protein sequencing and DNA sequencing

Section B: Protein StructureYang Xu, College of Life Sciences That’s all for Section B