Introduction to Molecular Biology zMolecular biology is interdisciplinary (biochemistry, genetics, cell biology) zImpact of genome projects (human, bacteria, fungi, plants, etc.)….”postgenomics era” zIntegration with other fields (e.g. computer science) leading to interdisciplinary career paths (Bioinformatics) Molecular Biology syllabus web siteweb site
Model Organisms
Central Dogma zDNA is transcribed to mRNA zmRNA is translated to protein
Proteins play many roles
Lecture 1 Protein Structure and Function Reading: Chapters 1-3
Protein Structure & Function -protein structure -protein purification & analysis -protein structure determination
Protein structure determines function zProteins are single, unbranched chains of amino acid monomers zThere are 20 different amino acids zA protein’s amino acid sequence determines its three-dimensional structure (conformation) zIn turn, a protein’s structure determines the function of that protein
Copyright (c) by W. H. Freeman and Company All amino acids have the same general structure but the side chain (R group) of each is different
Fig 3-2
Copyright (c) by W. H. Freeman and Company Hydrophilic amino acids Figure 2-13
Copyright (c) by W. H. Freeman and Company Hydrophobic & “special” amino acids Figure 2-13
Copyright (c) by W. H. Freeman and Company Peptide bonds connect amino acids into linear chains
Amino acids are the repeating units in proteins, but it is the 3-D protein structure that underlies function. How is 3-D structure obtained?
Copyright (c) by W. H. Freeman and Company Four levels of structure determine the shape of proteins zPrimary: the linear sequence of amino acids zSecondary: the localized organization of parts of a polypeptide chain (e.g., the helix or sheet) zTertiary: the overall, three-dimensional arrangement of the polypeptide chain zQuaternary: the association of two or more polypeptides into a multi-subunit complex
Secondary structure: the helix Figure 3-3
Copyright (c) by W. H. Freeman and Company Secondary structure: the beta sheet
Copyright (c) by W. H. Freeman and Company Motifs are regular combinations of secondary structures A coiled coil motif is formed by two or more helices wound around one another
Copyright (c) by W. H. Freeman and Company Other examples of motifs
Copyright (c) by W. H. Freeman and Company Primary and secondary structure in hemagglutinin
Copyright (c) by W. H. Freeman and Company Tertiary and quaternary structure in hemagglutinin
Sequence homology suggests functional and evolutionary relationships between proteins Figure 3-10
Folding, modification, & degradation of proteins zA newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein zAll molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule
Copyright (c) by W. H. Freeman and Company The information for protein folding is encoded in the sequence
Copyright (c) by W. H. Freeman and Company Folding of proteins in vivo is promoted by chaperones Figure 3-15
Copyright (c) by W. H. Freeman and Company Chemical modifications and processing alter the biological activity of proteins
Copyright (c) by W. H. Freeman and Company Protein degradation via the ubiquitin-mediated pathway Figure 3-13 Cells contain several other pathways for protein degradation in addition to this pathway
Copyright (c) by W. H. Freeman and Company Aberrantly folded proteins are implicated is slowly developing diseases An amyloid plaque in Alzheimer’s disease is a tangle of protein filaments
Copyright (c) by W. H. Freeman and Company Functional design of proteins zProtein function generally involves conformational changes zProteins are designed to bind a range of molecules (ligands) yBinding is characterized by two properties: affinity and specificity zAntibodies exhibit precise ligand-binding specificity zEnzymes are highly efficient and specific catalysts yAn enzyme’s active site binds substrates and carries out catalysis
Copyright (c) by W. H. Freeman and Company Kinetics of an enzymatic reaction are described by V max and K m
Copyright (c) by W. H. Freeman and Company Mechanisms that regulate protein function zAllosteric transitions yRelease of catalytic subunits, active inactive states, cooperative binding of ligands zPhosphorylation dephosphorylation zProteolytic activation zCompartmentalization
Copyright (c) by W. H. Freeman and Company Purifying, detecting, and characterizing proteins zA protein must be purified to determine its structure and mechanism of action zMolecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties
Copyright (c) by W. H. Freeman and Company Integral membrane proteins can be solubilized by non-ionic detergents
Copyright (c) by W. H. Freeman and Company Centrifugation can separate molecules that differ in mass or density
Copyright (c) by W. H. Freeman and Company Electrophoresis separates molecules according to their charge:mass ratio SDS-polyacrylamide gel electrophoresis
Copyright (c) by W. H. Freeman and Company Two-dimensional electrophoresis separates molecules according to their charge and their mass
Copyright (c) by W. H. Freeman and Company Separation of proteins by size: gel filtration chromatography
Copyright (c) by W. H. Freeman and Company Separation of proteins by charge: ion exchange chromatography
Copyright (c) by W. H. Freeman and Company Separation of proteins by specific binding to another molecule: affinity chromatography
Copyright (c) by W. H. Freeman and Company Highly specific enzymes and antibody assays can detect individual proteins
Copyright (c) by W. H. Freeman and Company Protein primary structure can be determined by chemical methods and from gene sequences Edman degradation
Copyright (c) by W. H. Freeman and Company Time-of-flight mass spectrometry measures the mass of proteins and peptides
Copyright (c) by W. H. Freeman and Company X-ray crystallography is used to determine protein structure Other techniques such as cryoelectron microscopy and NMR spectroscopy may be used to solve the structures of certain types of proteins