Page 1
Introduction to Molecular Biology
Molecular biology is interdisciplinary (biochemistry, genetics, cell biology)
Impact of genome projects (human, bacteria, fungi, plants, etc.)….”postgenomics era”
Integration with other fields (e.g. computer science) leading to interdisciplinary career paths (Bioinformatics)
Molecular Biology syllabus web site
Page 3
Central Dogma
DNA is transcribed to mRNA
mRNA is translated to protein
Page 4
Proteins play many roles
Page 5
Lecture 1
Protein Structure and Function
Reading: Chapters 1-3
Page 6
Protein Structure & Function
- protein structure
- protein purification & analysis
- protein structure determination
Page 7
Protein structure determines function
Proteins are single, unbranched chains of amino acid monomers
There are 20 different amino acidsA protein’s amino acid sequence
determines its three-dimensional structure (conformation)
In turn, a protein’s structure determines the function of that protein
Page 8
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
Page 9
Copyright (c) by W. H. Freeman and Company
Hydrophilic amino acids
Figure 2-13
Page 10
Copyright (c) by W. H. Freeman and Company
Hydrophobic & “special” amino acids
Figure 2-13
Page 11
Copyright (c) by W. H. Freeman and Company
Peptide bonds connect amino acids into linear chains
Page 13
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?
Page 14
Copyright (c) by W. H. Freeman and Company
Four levels of structure determine the shape of proteins
Primary: the linear sequence of amino acidsSecondary: the localized organization of parts
of a polypeptide chain (e.g., the helix or sheet)
Tertiary: the overall, three-dimensional arrangement of the polypeptide chain
Quaternary: the association of two or more polypeptides into a multi-subunit complex
Page 15
Secondary structure: the helix
Figure 3-4
The spiral is held by hydrogen bondsbetween nearly adjucent backbone O and H atoms
Page 16
Copyright (c) by W. H. Freeman and Company
Secondary structure: the beta sheet
Hydrogen bonds occur between backbone O and H of separate ajucent strands
Page 17
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 heliceswound around one another
Page 18
Copyright (c) by W. H. Freeman and Company
Other examples of motifs
Page 19
Tertiary structure // quaternary structure
hemagglutinin
Regions of proteins form domains: functional, topological or structural (like in case of HA)
Hydrophobic, hydrophylicinteractions anddisulfide bondshelp to keep the structure
The structure is stabilized by Interactions between domains
Page 20
Sequence homology suggests functional and evolutionary relationships between proteins
Figure 3-10
Page 21
Folding, modification, & degradation of proteins
A newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein
All molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule
Page 22
The information for protein folding is encoded in the sequence
Page 23
Folding of proteins in vivo is promoted by chaperones
Page 24
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
Page 25
Copyright (c) by W. H. Freeman and Company
Chemical modifications and processing alter the biological activity of proteins
Page 26
Copyright (c) by W. H. Freeman and Company
Protein degradation via the ubiquitin-mediated pathway
Cells contain several other pathways for protein degradation in addition to this pathway
Page 27
Copyright (c) by W. H. Freeman and Company
Functional design of proteins
Protein function generally involves conformational changes
Proteins are designed to bind a range of molecules (ligands) Binding is characterized by two properties: affinity
and specificityAntibodies exhibit precise ligand-binding specificityEnzymes are highly efficient and specific catalysts
An enzyme’s active site binds substrates and carries out catalysis
Page 28
Copyright (c) by W. H. Freeman and Company
Kinetics of an enzymatic reaction are described by Vmax and Km
Page 29
Copyright (c) by W. H. Freeman and Company
Mechanisms that regulate protein function
Allosteric transitions Release of catalytic subunits, active / inactive
states, cooperative binding of ligandsPhosphorylation / dephosphorylationProteolytic activationCompartmentalization
Page 30
Copyright (c) by W. H. Freeman and Company
Purifying, detecting, and characterizing proteins
A protein must be purified to determine its structure and mechanism of action
Molecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties
Page 31
Copyright (c) by W. H. Freeman and Company
Centrifugation can separate molecules that differ in mass or density
Page 32
Copyright (c) by W. H. Freeman and Company
Electrophoresis separates molecules according to their charge:mass ratio
SDS-polyacrylamidegel electrophoresis
Page 33
Copyright (c) by W. H. Freeman and Company
Two-dimensional electrophoresis separates molecules according to their charge and their mass
Page 34
Copyright (c) by W. H. Freeman and Company
Separation of proteins by size: gel filtration chromatography
Page 35
Copyright (c) by W. H. Freeman and Company
Separation of proteins by charge: ion exchange chromatography
Page 36
Copyright (c) by W. H. Freeman and Company
Separation of proteins by specific binding to another molecule: affinity chromatography
Page 37
Copyright (c) by W. H. Freeman and Company
Highly specific enzymes and antibody assays can detect individual proteins
Page 38
Copyright (c) by W. H. Freeman and Company
Time-of-flight mass spectrometry measures the mass of proteins and peptides
Page 39
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