cell bio ch 4 - The Proteins •  Enzymes...

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Unformatted text preview: 1/22/12 The Proteins •  Enzymes ( phosphofructokinase ) Essential Cell Biology Third Edition •  Structural Proteins ( collagen ) •  Transporter Proteins ( cystic fibrosis transmembrane regulator ) •  Motor Proteins ( myosin ) •  Storage Proteins ( ferritin ) Chapter 4 Protein Structure and Function Copyright © Garland Science 2010 •  Signal Proteins ( insulin ) •  Receptor Proteins ( rhodopsin ) •  Gene Regulatory Proteins ( lac repressor ) •  “Special Purpose” Proteins ( green fluorescent protein ) Protein Structure Primary structure (amino acid sequence) Protein folding Elements of secondary structure (α helix / β sheet) Tertiary structure (domains & modules) Quaternary structure/Multi-subunit protein complexes Self assembly of protein complexes 4o Multimeric Complex Protein Domains 3o (functional / structural) 2o Secondary Structural Elements (α helix / β sheet) 1o Amino Acid Sequence 1 1/22/12 Proteins are polypeptides made from the 20 amino acids Proteins are polypeptides made from the 20 amino acids •  It’s the side chains that give an amino acid it’s unique properties. The Amino Acids •  •  •  The Amino Acids Aspartic acid also called aspartate Glutamic acid also called glutamate Glycine can often be considered polar since it can participate in H-bonding. Figure 4-3 Essential Cell Biology (© Garland Science 2010) 2 1/22/12 Always write AA sequence from N to C termini 5 AA “Polypeptide” (1) (2) (3) (4) Polypeptides = Peptides (small) & Proteins (large) “Polypeptide” Primary Structure A source of tremendous structural diversity 4o 20 Common Amino Acids (AA) à༎ 20n possible peptides of n AA Multimeric Complex Protein Example 1 Domains 8 AA peptides are common signaling molecules There are 208 = 25,600,000,000 possible unique 8 AA peptides 3o (functional / structural) 2o Secondary Structural Elements Example 2 Average protein ~ 300 AA à༎ 20300 = 10390 > > # atoms in universe ! However, not all amino acids are equally represented in proteins (α helix / β sheet) C + W + M = 5% L + S + K + E = 32% 1o Amino Acid Sequence 3 1/22/12 Amino Acids involved in ligand binding and catalysis Figure 4-3 Essential Cell Biology (© Garland Science 2010) Amino acid side chains available for phosphorylation Figure 4-3 Essential Cell Biology (© Garland Science 2010) Amino acid side chains available for glycosylation Figure 4-3 Essential Cell Biology (© Garland Science 2010) Figure 3-3a Molecular Biology of the Cell (© Garland Science 2008) 4 1/22/12 Somehow, a protein manages to fold into a single stable conformation/shape Can take on many shapes Noncovalent bonds help proteins fold •  A few exceptions. •  Regardless, this infers that the final 3D shape of a protein is determined mostly by its AA sequence. •  However, folding is often aided by molecular chaperones. More efficient and reliable. Figure 4-7 Essential Cell Biology (© Garland Science 2010) So do hydrophobic “interactions” This particular case also resembles a hydrophobic interaction (talk about next). Figure 4-4 Essential Cell Biology (© Garland Science 2010) Proteins come in many different shapes and sizes •  While the AA sequence determines the 3D shape, we still can not determine the final 3D shape by knowing the sequence alone. •  Need X-ray crystallography or NMR. Figure 4-5 Essential Cell Biology (© Garland Science 2010) Figure 4-9 Essential Cell Biology (© Garland Science 2010) 5 1/22/12 Molecular chaperones (proteins) assist in the proper folding of substrate proteins in the ER lumen Molecular chaperones bind to non-polar amino acid side chains to prevent misfolding during translation This is an iterative process 1. In the presence of ATP, chaperones bind as homodimers 2. ATP hydrolysis is required for dissociation of the chaperone Figure 6-85 Molecular Biology of the Cell (© Garland Science 2008) Ubiquitin 76 AA “protein” Targets proteins for destruction Figure 6-92a Molecular Biology of the Cell (© Garland Science 2008) Figure 6-93 Molecular Biology of the Cell (© Garland Science 2008) 6 1/22/12 Poly-ubiquitination targets protein for degradation in the 20S proteasome Incorrect protein folding can lead to disease (esp neurodegenerative diseases) Huntington’s Alzheimer’s Creutzfeldt-Jacob Mad Cow Ubiquitin is depolymerized and recycled Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008) 4o Multimeric Complex Protein Domains 3o (functional / structural) 2o Secondary Structural Elements Secondary structures give local conformational stability •  The 2 most common secondary structures. •  Results from H-bonds between N-H and C=O groups in the polypeptide backbone. Since no side chains involved, these can be generated by many different AA sequences •  Abundant in skin, hair, nails, horns. •  Abundant in silk. (α helix / β sheet) 1o Amino Acid Sequence 7 1/22/12 α-helix •  In α-helix, the N-H of every peptide bond is hydrogen-bonded to the C=O of a neighboring peptide bond 4 AAs away on same chain (get a complete turn every 3.6 AAs). Figure 4-10a–c Essential Cell Biology (© Garland Science 2010) Figure 4-11 Essential Cell Biology (© Garland Science 2010) α-helices are abundant in proteins located in cell membranes Multiple α-helices can wrap around one another to form extra stable structures •  Good way to anchor protein. Ex. α-keratin (skin) and myosin (muscle) •  ~ 20 AAs are needed to span a lipid bilayer. •  Can often be predicted from sequence alone. Movie 4.3 Figure 4-12 Essential Cell Biology (© Garland Science 2010) Figure 4-13 Essential Cell Biology (© Garland Science 2010) 8 1/22/12 β-sheets are rigid structures β-sheets come in 2 flavors antiparallel Ex. silk Movie 4.4 parallel •  In β-sheet, the individual polypeptide chains (strands) in the sheet are held together by H-bonding between peptide bonds in different strands (or at least far away in same chain) and the AA side chains in each strand stick out alternatively above and below the sheet. 4o Multimeric Complex Protein Domains 3o (functional / structural) 2o Figure 4-14 Essential Cell Biology (© Garland Science 2010) Most proteins are formed from multiple domains •  Domains are stable regions of a protein consisting of groups of secondary structural elements. •  A domain often is functional motif of a protein that retains its properties in isolation. •  Domains often linked via unstructured regions. Secondary Structural Elements (α helix / β sheet) 1o Amino Acid Sequence 9 1/22/12 Modular Mix-N-Match gives rise to proteins with new combinations of properties Proteins are classified as families •  Tertiary structure is preserved within families •  Have conserved regions 2 members of the serine protease family of proteins Figure 3-15 Molecular Biology of the Cell (© Garland Science 2008) Viewing proteins 4o Multimeric Complex (more than 1 polypeptide) Protein Domains 3o (functional / structural) 2o Secondary Structural Elements Movie 4.1 (α helix / β sheet) 1o Amino Acid Sequence 10 1/22/12 Large proteins often contain multiple polypeptide chains tetramer dimer same polypeptide Proteins can assemble into complex repeating structures tetramer Common in extracellular matrix, actin, collagen, elastin, intermediate filaments. different polypeptides •  The same weak noncovalent bonds that allow a polypeptide to fold also allow protein-protein interactions via binding sites. •  Each polypeptide chain in multipolypeptide protein is called a subunit. •  Shown above are examples of globular proteins. Figure 4-21 Essential Cell Biology (© Garland Science 2010) Single protein subunits can pack to form a filament, a tube or a spherical shell Figure 4-23 Essential Cell Biology (© Garland Science 2010) Figure 4-24 Essential Cell Biology (© Garland Science 2010) 11 1/22/12 Many proteins have long fibrous shapes (Ex. collagen) •  Actin is a rigid, “rod-like” assemblage of globular subunits. Protein Function •  Proteins bind to other molecules. (Affinity measured as Km). •  Anything that binds to a protein is referred to as a ligand. •  This binding is governed by weak noncovalent interactions. Thus, to selectively bind a ligand, a protein’s binding site must make many interactions with a ligand. Disulfide bonds are covalent bonds between cysteine residues •  Extracellular proteins often use these. (ex. curly hair) •  Typically not found in cytosol (milder than extracellular). •  Since they are covalent, they are a major stabilizing force. Binding sites allow a protein to interact with specific ligands Figure 4-28 Essential Cell Biology (© Garland Science 2010) 12 1/22/12 Binding is highly developed in antibodies (immunoglobulins) Protein Function •  Proteins can be enzymes. Often work in tandem to form pathways •  Enzymes bind to one or more ligands, called substrates, and convert them into chemically modified products, doing this rapidly over and over again without themselves being changed. Movie 4.7 •  Heavily used in research •  Critical for self vs nonself recognition Enzyme example--lysozyme Chemical closeup--lysozyme •  Lysozyme can distort chemical bonds in such a way (transition state) as to reduce the activation energy needed for hydrolysis. Movie 4.8 13 1/22/12 Enzyme can promote catalysis in several ways Pharmacology (Ex. lysozyme) •  Most drugs target enzymes. •  Specifically, most drugs target “receptors”. Figure 4-32 Essential Cell Biology (© Garland Science 2010) Prosthetic groups extend the capabilities of enzymes Protein regulation •  Substrate/product concentration (Le Chatelier’s principle) Senses light heme •  Compartmentalization/location •  At the enzyme itself •  Happens by altering the enzyme’s shape and therefore its function cis- retinal •  Our bodies can’t make, need from our diet—vitamin A red Hemoglobin in your blood contains this. Low iron à༎ anemia 14 1/22/12 Feedback regulation is a common type of control Regulation often occurs via allostery •  Occurs when a molecule other than the substrate binds an enzyme and regulates its activity. •  Can be either negative (bottom left and middle) or positive (bottom right). •  Common in metabolism. •  Binding at one site alters the activity of another part of the enzyme. •  Allosteric regulators induce conformational changes. Ex. Aspartate transcarbamoylase Figure 4-36 Essential Cell Biology (© Garland Science 2010) aspartate carbamoyltransferase: an allosteric enzyme complex controlling pyrimidine biosynthesis Reversible phosphorylation is a major mechanism of signal transduction •  Can occur on serine, threonine or tyrosine. CTP •  Requires ATP. •  Can either activate or inhibit proteins (below). 12 subunits C6R6 Figure 4-38a Essential Cell Biology (© Garland Science 2010) 15 1/22/12 GTP activates “small GTPase/GTP-binding” signal proteins “G-proteins” Nucleotide hydrolysis is also used by motor proteins to produce movement in cells Ex. myosin “runs” along actin in your muscles. •  Example: Movie 4.10 •  Largest drug target class is the GPCRs No ATP = aimless wandering Figure 4-39 Essential Cell Biology (© Garland Science 2010) Proteins often form large complexes that function as protein machines Covalent modifications to proteins control many different aspects of their activity both directly and indirectly (the protein code) •  Each central process in a cell (ex. DNA replication, protein synthesis, etc) is catalyzed by a highly coordinated, linked set of many proteins. •  In most of these protein machines the hydrolysis of ATP or GTP drives an ordered series of conformational changes. Useful for successive reactions. Greater efficiency!! •  Each modification sometimes like creating a new protein •  Huge area of future research (20 well known modification sites) Figure 4-43 Essential Cell Biology (© Garland Science 2010) 16 1/22/12 PANEL 4–4 164 Some proteins integrate signals through subunit interactions PANEL 4–4 164 BREAKING CELLS AND TISSUES BREAKING CELLS AND TISSUES The first step in the purification of most proteins is to disrupt tissues and cells in a controlled fashion. Cell breakage and initial fractionation of cell extracts Studying proteins Cell breakage and initial fractionation of cell extracts The first step in the purification of most proteins soup (called The resulting thick is to disrupt tissues and cells in a a homogenate or an extract) controlled fashion. Using gentle mechanical procedures, called homogenization, the plasma membranes of cells can be ruptured so that the cell contents are released. Four commonly used procedures are shown here. Using gentle mechanical procedures, called homogenization, the plasma membranes of cells can be ruptured so that the cell contents are released. Four commonly used procedures are shown here. contains large and small molecules from the cytosol, such as enzymes, ribosomes, and metabolites, as well as all of the membrane-enclosed organelles. Need pure protein to study 1 Break cells with high-frequency sound. 1 Break cells with high-frequency sound. 2 Use a mild detergent to make holes in the plasma membrane. 3 Force cells through a small hole using high pressure. 3 Force cells through a small hole using high pressure. When carefully conducted, homogenization leaves most of the membrane-enclosed organelles intact. 4 Shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel. THE CENTRIFUGE Cyclin-dependent protein kinase armored chamber 4 Shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel. When carefully conducted, homogenization leaves most of the membrane-enclosed organelles intact. swinging-arm rotor THE CENTRIFUGE armored chamber swinging-arm rotor 2 Use a mild detergent to make holes in the plasma membrane. •  First need to break open/lyse cells cell suspension or tissue cell suspension or tissue The resulting thick soup (called a homogenate or an extract) contains large and small molecules from the cytosol, such as enzymes, ribosomes, and metabolites, as well as all of the membrane-enclosed organelles. centrifugal force tube sedimenting material metal bucket CENTRIFUGATION centrifugal force tube Many cell fractionations are done in a second type of rotor, a swinging-arm rotor. sedimenting material metal bucket CENTRIFUGATION •  Then typically fractionate cell extracts using centrifugation Many cell fractionations are done in a second type of rotor, a swinging-arm rotor. fixedangle rotor CELL HOMOGENATE before centrifugation vacuum CELL HOMOGENATE before centrifugation The metal buckets that hold the tubes are free to swing outward as the rotor turns. The metal buckets that hold the tubes are free to swing outward as the rotor turns. SUPERNATANT smaller and less dense components CENTRIFUGATION PELLET larger and more dense components SUPERNATANT smaller and less refrigeration dense components motor CENTRIFUGATION BEFORE refrigeration fixedangle rotor BEFORE AFTER vacuum Centrifugation is the most widely used procedure to separate a PELLET homogenate into different parts, or fractions. The homogenate is larger and more placed in test tubes and rotated at high speed in a centrifuge or dense components ultracentrifuge. Present-day ultracentrifuges rotate at speeds up AFTER to 100,000 revolutions per minute and produce enormous forces, as high as 600,000 times gravity. Such speeds require centrifuge chambers to be refrigerated and evacuated so that friction does not heat up the homogenate. The centrifuge is surrounded by thick armor plating, because an unbalanced rotor can shatter with an explosive release of energy. A fixed-angle rotor can hold larger volumes than a swinging-arm rotor, but the pellet forms less evenly. motor PANEL 4–5 166 Centrifugation is the most widely used procedure to separate a homogenate into different parts, or fractions. The homogenate is placed in test tubes and rotated at high speed in a centrifuge or ultracentrifuge. Present-day ultracentrifuges rotate at speeds up to 100,000 revolutions per minute and produce enormous forces, as high as 600,000 times gravity. PROTEIN SEPARATION _ + + + _ + _ + + Such speeds require centrifuge chambers to be refrigerated and evacuated so that friction does not heat up the homogenate. The centrifuge is surrounded by thick armor plating, because an unbalanced rotor can shatter with an explosive release of energy. A fixed-angle rotor can hold larger volumes than a swinging-arm rotor, but the pellet forms less evenly. Protein separation by chromatography COLUMN CHROMATOGRAPHY _ _ _ _ Proteins are often fractionated by column chromatography. A mixture of proteins in solution is applied to the top of a cylindrical column filled with a permeable solid matrix immersed in solvent. A large amount of solvent is then pumped through the column. Because different proteins are retarded to different extents by their interaction with the matrix, they can be collected separately as they flow out from the bottom. According to the choice of matrix, proteins can be separated according to their charge, hydrophobicity, size, or ability to bind to particular chemical groups (see below ). sample applied + solvent continuously applied to the top of column from a large reservoir of solvent Proteins are very diverse. They differ in size, shape, charge, hydrophobicity, and their affinity for other molecules. All of these properties can be exploited to separate them from one another so that they can be studied individually. THREE KINDS OF CHROMATOGRAPHY Studying proteins •  Common approaches to isolate proteins: •  Chromatography (following which test different fractions to see which has protein of interest) •  Size •  Charge •  Binding •  Electrophoresis •  Size •  Charge Although the material used to form the matrix for column chromatography varies, it is usually packed in the column in the form of small beads. A typical protein purification strategy might employ in turn each of the three kinds of matrix described below, with a final protein purification of up to 10,000-fold. Purity can easily be assessed by gel electrophoresis (Panel 4–6). solid matrix porous plug Chromatography test tube time fractionated molecules eluted and collected charge size binding solvent flow solvent flow solvent flow + + + ++ + + + + +++ + + + + + + + + + ++ + + + ++ positively charged bead + + +++ + + + + + +++ + + + + + bound negatively charged molecule free positively charged molecule (A) ION-EXCHANGE CHROMATOGRAPHY Ion-exchange columns are packed with small beads carrying either positive or negative charges that retard proteins of the opposite charge. The association between a protein and the matrix depends on the pH and ionic strength of the solution passing down the column. These can be varied in a controlled way to achieve an effective separation. porous beads small molecules retarded large molecules unretarded bead with covalently attached substrate bound enzyme molecule other proteins pass through (B) GEL-FILTRATION CHROMATOGRAPHY (C) AFFINITY CHROMATOGRAPHY Gel-filtration columns separate proteins according to their size. The matrix consists of tiny porous beads. Protein molecules that are small enough to enter the holes in the beads are delayed and travel more slowly through the column. Proteins that cannot enter the beads are washed out of the column first. Such columns also allow an estimate of protein size. Affinity columns contain a matrix covalently coupled to a molecule that interacts specifically with the protein of interest (e.g., an antibody, or an enzyme substrate). Proteins that bind specifically to such a column can subsequently be released by a pH change or by concentrated salt solutions, and they emerge highly purified (see also Figure 4–49). 17 1/22/12 Electrophoresis PANEL 4–6 167 Protein separation by electrophoresis GEL ELECTROPHORESIS sample loaded onto gel by pipette cathode plastic casing The detergent sodium dodecyl sulfate (SDS) is used to solubilize proteins for SDS polyacrylamidegel electrophoresis. protein with two subunits, A and B, joined by a disulfide bridge CH3 CH2 CH2 A CH2 single subunit protein B C S-S CH2 CH2 CH2 HEATED WITH SDS AND MERCAPTOETHANOL CH2 __ __ _ _ __ _ __ _ __ __ _ _ __ _ __ _ _ _ _ ___ _ _ __ ___ _ __ _ _ _ _ _ __ _ _ __ _ _ __ _ __ __ _ _ _ _ _SH__ __ _ _ _ _ ___ _ _ _ _ _ __ __ __ _ _ __ ___ __ _ __ _ __ _ _ _ _ _ __ __ _ _ __ __ __ __ HS _ _ _ __ _ __ _ ___ _ _ _ _ _ _ __ _ __ __ _ _ _ _ __ _ __ __ _ _ ___ _ _ negatively __ _ _ _ _ _ _ __ _ ___ _ charged SDS _ _ __ __ C _ _ _ __ _ _ __ molecules A B CH2 buffer CH2 + anode gel CH2 CH2 O O buffer ISOELECTRIC FOCUSING For any protein there is a characteristic pH, called the isoelectric point, at which the protein has no net charge and therefore will not move in an electric field. In isoelectric focusing, proteins are electrophoresed in a narrow tube of polyacrylamide gel in which a pH gradient is established by a mixture of special buffers. Each protein moves to a point in the gradient that corresponds to its isoelectric point and stays there. stable pH gradient 9 8 7 6 5 4 At low pH, the protein is positively charged. ++ _ +_ _+ + + _+_ +_ _+ + _+_ +_ _+ + + _ +_ _ _ __+ POLYACRYLAMIDE-GEL ELECTROPHORESIS Na + B SDS polyacrylamide-gel electrophoresis (SDS-PAGE) Individual polypeptide chains form a C complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS–protein complex through a slab of porous polyacrylamide gel. The A apparatus used for this electrophoresis technique is shown above (left ). A reducing agent (mercaptoethanol) is usually added to break any –S–S– linkages in or between proteins. Under these conditions, proteins migrate at a rate that reflects their molecular weight. + slab of polyacrylamide gel TWO-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS ..Western Blot Complex mixtures of proteins cannot be resolved well on one-dimensional gels, but two-dimensional gel electrophoresis, combining two different separation methods, can be used to resolve more than 1000 proteins in a two-dimensional protein map. In the first step, native proteins are separated in a narrow gel on the basis of their intrinsic charge using isoelectric focusing (see left ). In the second step, this gel is placed on top of a gel slab, and the proteins are subjected to SDS-PAGE (see above ) in a direction perpendicular to that used in the first step. Each protein migrates to form a discrete spot. + +++ + + +++ ___ _ _ ___ At high pH, the protein is negatively charged. O SDS The protein shown here has an isoelectric pH of 6.5. All the proteins in an E. coli bacterial cell are separated in this 2-D gel, in which each spot corresponds to a different polypeptide chain. They are separated according to their isoelectric point from left to right and to their molecular weight from top to bottom. (Courtesy of Patrick O'Farrell.) basic SDS migration (mol. wt. x 10–3) 10 S O When an electric field is applied to a solution containing protein molecules, the molecules will migrate in a direction and at a speed that reflects their size and net charge. This forms the basis of the technique called electrophoresis. stable pH gradient acidic 100 50 25 SDS-PAGE SDS-PAGE •  The actual bands are equal in size, but the proteins within each band are of different sizes. 18 1/22/12 Studying proteins •  After purify protein •  Digestion and sequencing à༎ 1st structure •  X-ray crystallography and NMR à༎ 3D structure (2nd-4th structure) •  Check function in vitro or in vivo (this is relative) •  Can often grow cells in culture •  Cheap •  Easy to manipulate •  Some drawbacks 19 ...
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This note was uploaded on 01/26/2012 for the course BIOL 4374 taught by Professor Staff during the Spring '08 term at University of Houston.

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