Chapter 3. Protein Structure and Function

Transcription

1 Chapter 3 Protein Structure and Function

2 Broad functional classes So Proteins have structure and function... Fine! -Why do we care to know more???? Understanding functional architechture gives us POWER to: Diagnose reasons for diseases Create modifying drugs Engineer our own designerproteins

3 Protein structure determines function DNA (mrna) Translation: Translation into 3D structure: Modifications: Chemical modification of aminoacids Interaction with other molecules Proteolytic cleavage (Location) 3D structure determines function: New 3D structure New function Proteins are single, unbranched chains of amino acid monomers There are 20 different amino acids The amino acid sidechains in a peptide can become modified, extending the functional repetoire of aminoacids to more than hundred different amino acids. A protein s amino acid sequence determines its three-dimensional structure (conformation) In turn, a protein s structure determines the function of that protein Conformation (=function) is dynamically regulated in several different ways

4 All amino acids have the same general structure but the side chain (R group) of each is different Cα R: Hydrophilic: Basic Acidic Non-charged Hydrophobic Special

5 Hydrophilic amino acids

6 Hydrophobic and special amino acids

7 Peptide bonds connect amino acids into linear chains Backbone Side-chains

8 Side chain modifications change the chemical (functional) properties of proteins Acetylation Phosphorylation Hydroxylation Methylation Carboxylation => Expanding the repetoire of existing amino acid side-chains to > 100 variations! Glycosylation Ubiquitylation

9 Four levels of structure determine the shape of proteins Primary: the linear sequence of amino acids peptide bonds Secondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet) backbone hydrogen bonds Tertiary: the overall, threedimensional arrangement of the polypeptide chain hydrophobic interactions, hydrogen bonds (non-covalent bonds in generel) And sulfur-bridges Quaternary: the association of two or more polypeptides into a multi-subunit complex Cys-Cys sulfur-bridges

10 Primary and secondary structure (example: hemagglutinin) β-strand α-helix

11 Secondary structure α Helix β Sheet β (U)-turn

12 Motifs are regular combinations of secondary structures. Motifs form Domains Three examples of Motifs forming different types of DNA-binding domains

13 Structural and functional domains are modules of tertiary structure Each of these proteins contain the EGF globular domain. - But each of these proteins have a different function Globular domain Tertiary structure

14 Different graphical representations of the same protein (tertiary structure)

15 Quaternary structure

16 Sequence homology suggests functional and evolutionary relationships between proteins When the stucture of a newly discovered protein is known, comparison to other proteins across species can help predict function

17 Folding, modification, and degradation of proteins The life of a protein can briefly be described as: synthesis, folding, modification, function, degradation. 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 Most proteins have a limited lifespan before they are degraded (turn-over time)

18 The information for protein folding is encoded in the sequence

19 Folding of proteins in vivo is promoted by chaperones Large proteins with much secondary structure may require assited folding to avoid aggregation of unfolded protein - Chaperones and chaperonins prevent aggregation of unfolded protein

20 Protein degradation via the ubiquitin-mediated pathway acetylation Cells contain several other pathways for protein degradation in addition to this pathway

21 Aberrantly folded proteins are implicated in slowly developing diseases An amyloid plaque in Alzheimer s disease is a tangle of protein filaments Figure 3-19

22 Functional design of proteins Protein function often involves conformational changes Proteins are designed to bind a range of molecules (ligands) Binding is characterized by two properties: affinity and specificity Antibodies and enzymes exhibit precise ligand/substratebinding specificity But can have variable affinities Enzymes are highly efficient and specific catalysts An enzyme s active site binds substrates(ligands) and carries out catalysis

23 Enzymes have high substrate affinity sites and catalytic sites NB: specificity versus affinity!

24 Kinetics of an enzymatic reaction are described by V max and K m

25 Kinetics of an enzymatic reaction are described by V max and K m

26 Mechanisms that regulate protein activity Allosteric transitions Release of catalytic subunits, active inactive states, cooperative binding of ligands Chemical modification: Phosphorylation, acetylation etc. dephosphorylation, deacetylation etc. Proteolytic activation Compartmentalization

27 Regulated function Conformational changes induced by Ca 2+ binding to calmodulin Cooperative binding of calcium: binding of one calcium enhances the affinity for the next calcium When 4 calcium are bound a major allosteric conformational change occurs A switch protein because this effect in turn regulates other proteins bound by the compact calmodulin

28 Regulated function Cooperative binding of ligands Sigmoidal curve indicates cooperative binding (of ligands, substrates, ca ions) in contrast to standard Michaelis-Menten Kinetics

29 Regulated function Chemical modification Example: Phosphorylation dephosphorylation

30 Regulated function Proteolytic cleavage of chymotrypsinogen to produce active chymotrypsin

31 Regulated function Compartmentalization Example:Membrane proteins Each cell membrane has a set of specific membrane proteins that allows the membrane to carry out its distinctive activities Membrane proteins are either integral or peripheral Integral transmembrane proteins contain one or more transmembrane α helices Other integral proteins are anchored to the membrane by covalently attached hydrocarbon chains Peripheral proteins are associated with membranes through interactions with integral proteins

32 Regulated function Schematic of membrane proteins in a lipid bilayer

33 Regulated function Glycophorin: an example transmembrane protein Figure 3-33

34 Regulated function Anchoring of integral proteins to the plasma membrane by hydrocarbon chains

35 Now that you KNOW the basic principles of protein structure and function you can UNDERSTAND: Protein and Proteome Analytical techniques

36 Purifying, detecting, and characterizing proteins A protein must be purified to determine its structure and mechanism of action Detecting known proteins can be usefull for diagnostic purposes Molecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties (size, mass, density, polarity, affinity...) Elementary toolbox includes: centrifugation, electrophoresis, liquid chromatography (LC), spectrometry, ionization/radiation. -applied in various advanced forms and combinations.

37 Centrifugation can separate molecules that differ in mass or density

38 Electrophoresis separates molecules according to their charge:mass ratio SDS-polyacrylamide gel electrophoresis 1 : 3.6 Even coating of proteins allows even charge distribution -> larger mass = higher total charge

39 Two-dimensional electrophoresis separates molecules according to their charge and their mass

40 Separation of proteins by size: gel filtration chromatography (LC) Add mobile phase: buffer Stationary phase:

41 Separation of proteins by charge: ion exchange chromatography Add mobile phase: low salt buffer Add mobile phase: high salt buffer Stationary phase: Also: Reversed-phase LC: separation by hydrophobicity Stationary phase: non-polar, Mobile phase: moderately polar

42 Separation of proteins by specific binding to another molecule: affinity chromatography

43 Highly specific enzymes and antibody assays can detect individual proteins Immunoblot (= Western Blot) based on affinity

44 Genomics, the analysis of complex protein mixtures Genome databases allow prediction of genes -> protein primary structure Each protein can be fragmented into peptides which are composed of aa s. Each aa has a unique mass to charge ratio at a given ph Each protein therefore has a unique peptide-fingerprint Technique: proteins->peptides->mass/charge ratio measurement -> compare against whole proteome (genome based) database -> identify proteins

45 Time-of-flight mass spectrometry measures the mass of proteins and peptides Matrix-Assisted-Laser-Desorption/Ionization Time-offlight mass spectrometry (MALDI-TOF MS)

46 Example of a proteome analysis workflow Cell/tissue of interest Isolate organelles (fractionation) Confirm organelle-specific proteins Subfractionate, detect peptides, identify corresponding proteins

47 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

48 After completion of this lecture you should be able to: Protein structure and function Draw the generel structure of amino acids Draw the generel structure of proteins (including N and C-terminal, alpha-carbons, R-groups, and peptide bonds) Know the 20 generel amino acids and how they are characterized (basic, acid, etc...) Understand that the properties (physical and chemical) of proteins can change with altered ph, temperature, hydrophobicity of the surroundings Define primary, secondary, tertiary, and quaternary structure of proteins. And how they influence conformation, hence function, of proteins Explain what a protein subunit means Explain how altered conformation (and function) can be achieved by sidechain modification of proteins. Mention the various types of sidechain modifications Understand that sidechain modification expands the actual repetoire of amino acids to more than 100 variations Know that most often correct protein folding is attained by chaperone-assisted folding Know that protein-degradation is often achieved through protein modification signaling degradation. Beispiel: ubiquitylation Explain the term: protein turn-over time Understand that incorrect protein folding can be detrimental and lead to aggrevating (serious) disease Mention and briefly describe the 4 major Mechanisms that regulate protein activity (Allosteric transitions, Chemical modification, Proteolytic activation, and Compartmentalization) Know what coordinated ligand-binding means

49 After completion of this lecture you should be able to: Protein and Proteome analytical techniques Explain that the elementary properties of proteins makes each protein/peptide uniqe and how this can be used to separate and purify proteins Mention the elementary biotechnical tools applied in protein analysis Explain the term Proteome Describe: Centrifugation: separation by mass or density Electrohopresis: separation by mass/charge (SDS-based) or by charge and then mass (2D-electrophoresis) Including: how SDS gives protein an even size-related charge Liquid chromatography (LC) Gel filtration: separation by size Ion exchange chromatography: separation by charge Reversed phase chromatography: separation by hydrophobicity (charge) Affinity chromatography: separation by affinity to known ligand (eg. Antibody) Immunoblot/Western blot: combination of electrohoresis and affinity -> allows detection of specific protein within protein mix, for example in a patient plasma sample or a cancer biopsy. Mass Spectrometry: Identifying proteins based on splitting them into peptides, each of which has a unique physical/chemical idenitiy, which can be found in a proteome database. including the basic principle of Time-of-flight Identification of many (often hundreds) proteins in a complex mixture (proteomics): how sequential combination (workflow) of the above mentioned techniques allow specific identification of for example all proteins contained in an organelle X-Ray Chrystallography: the basic concept of how X-rays become differentially diffracted by the atoms of chrystallized protein. Most important: that this technique is used for determining protein structure at atomic level.

50 Next week: Nucleic Acids, the Genetic Code, and the Synthesis of Macromolecules