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This 25 page Class Notes was uploaded by Amalachi Notetaker on Sunday October 9, 2016. The Class Notes belongs to CHEM3510 at University of Toledo taught by Bellizzi,J in Fall 2016. Since its upload, it has received 4 views. For similar materials see Biochemistry I in Natural Sciences and Mathematics at University of Toledo.
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Date Created: 10/09/16
Lecture 13 – UPDATED 19 Sept Protein Folding and Denaturation Protein Purification and Analysis CHEM 3510 Biochemistry I Dr. John Bellizzi Fall 2016 Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 2 Protein denaturation (unfolding) Denatured state – native structure is disrupted (to the extent that biological activity is lost) What causes denaturation? • Heat, extreme pH, organic solvents, detergents (e.g. SDS) • Chaotropes: Urea (8 M), Guanidinium chloride (guanidine hydrochloride) (6 M) Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 3 Protein folding is spontaneous and rapid • Under physiological conditions, proteins spontaneously adopt their native conformations. • Primary structure contains all the information necessary for 3D structure • Unfolded protein has many noncovalent interactions with water molecules • As it folds, it exchanges interactions with water for interactions with itself • Pairing of H-bond donors and acceptors, dispersion forces, hydrophobic effect • Evidence? Denatured proteins can spontaneously refold (renature) into their native conformations! Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 4 Protein folding is hierarchical Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 5 Gel Electrophoresis Gel (crosslinked polymer mesh) • Agarose or polyacrylamide • Molecular sieving – smaller molecules pass through mesh faster Electrophoresis • Charged particles (including proteins!) migrate in electric field Gels may be run under native conditions (protein remains in folded 3D conformation) or denaturing conditions (protein is unfolded). Most common denaturing protein gel = SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi6 SDS-P AGE Analysis of Proteins Sodium dodecyl sulfate - Detergent (amphipathic) Denatures (unfolds) proteins Coats denatured protein molecules with uniform negative charge Proteins will all migrate towards positive electrode Larger proteins migrate more slowly Used to analyze size, purity of proteins. After electrophoresis, gel must be stained to visualize proteins. Common stain – Coomassie Brilliant Blue Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi7 Using SDS-P AGE to determine M r Mobility of a protein on SDS-PAGE is proportional to the log of the M r Using a standard “ladder” of proteins of knonw Mr, a calibration curve can be made to determine the M rf another protein on the gel. Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 8 Isoelectric Focusing (IEF) Gel Electrophoresis • Gel mixture includes ampholytes (polyionic polymers) • Applied electric field leads to stable pH gradient in gel • Addition of sample and electrophoresis causes proteins to migrate according to pI (will move to region of gel where pH = pI) Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 9 Two-Dimensional Gel Electrophoresis Two separations using both techniques (IEF and SDS-PAGE) Allows resolution of complex mixtures of proteins (e.g. the proteome of an entire cell!) Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 10 Protein Purification Isolate and purify molecules of that protein from cellular extract Need to exploit differences between target protein and other macromolecules (e.g. charge, size, specific binding, solubility…) Multistep process: • Begin with crude cellular extract (soluble fraction of cell lysate) • Ammonium sulfate precipitation (salting out) • Chromatography Monitoring purification: • measure target protein/total protein • For enzymes – specific activity (catalytic activity/total protein) • Gel electrophoresis Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 11 Chromatography Separation based on the differential movement of solutes in a mobile (liquid) phase when passed through a stationary (porous solid) phase. Most protein chromatography is carried out in columns, with flow driven by gravity, centrifugation, or pumps. Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 12 Slides after this point will NOT be on Exam 1. Anything we don’t get to in Wednesday’s lecture will not be covered in class but you are still responsible for the material in the associated chapters. • X-ray crystallography and NMR for determining protein 3D structure pp. 134-135 • Protein Misfolding and Disease pp. 148-151 • Types of Protein Chromatography pp. 90-91 • Chemical synthesis of peptides pp. 102-104 • CD Spectroscopy p. • Intrinsically Disordered Proteins pp.141-142 • Folding Accessory Proteins pp. 146-148 Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 13 Protein Purification by Chromatography Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 14 Protein Purification by Chromatography Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 15 Protein 3D Structure Determination X-ray crystallography Purify protein Crystallize protein X-ray diffraction experiment Intensities/directions of scattered X- rays used to calculate electron density Build molecular model using knowledge of primary structure to fit the experimental electron density Works for any protein (or other molecule or complex), regardless of size… …as long as you can grow crystals of it! Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 16 Protein 3D Structure Determination Biomolecular Nuclear Magnetic Resonance Spectroscopy • Structures of purified proteins in solution (can be used to study dynamics as well as structure) • Two-dimensional NMR techniques: • Nuclear Overhauser Effect Spectroscopy (NOESY) • Measures distance between nonbonded atoms through space • Total Correlation Spectroscopy (TOCSY) • Measures distance between bonded atoms. No need to crystallize proteins, but only works well with small proteins. Generates ensemble of structures. Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 17 Diseases associated with misfolded proteins Amyloidoses • Misfolded/partially folded secreted proteins associate via β-sheets to form long amyloid fibril • Slow aggregation of insoluble fibrils in affected tissues • Pancreatic islet β-cells (insulin secretion) • Islet amyloid polypeptide (37 aa) can form amyloid deposits, lead to loss of β-cells and Type II diabetes. • Alzheimer’s Disease • Extracellular deposits of amyloid β-peptide • Derived from transmembrane segment of larger protein (amyloid β-precursor protein) • When cleaved, loses helical structure – forms amyloid fibril • Also amyloid-like aggregates of tau protein inside neurons Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 18 Neurological Diseases Caused By Aggregates of Misfolded Protein Alzheimer’s Disease Huntington’s Disease Parkinson’s Disease Prion Diseases Scrapie (sheep), chronic wasting disease (deer) Bovine spongiform encephalitis (BSE) – “Mad Cow disease” In humans: Kuru, inherited spongiform encephalopathies (Creutzfeldt-Jakob disease and Alper’s syndrome) • Prions (protein infectious only) C Sc • PrP (normal brain protein) → PrP (catalyzed by PrP )c Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 19 Chemical Synthesis of Peptides Peptides synthesized from C-terminus to N-terminus attached to solid support Challenges: • Need to make sure the only peptide bond formed is between the desired amino group and carboxylate • Need to activate carboxylate for condensation (carboxylate is not reactive for acyl substitution) • Efficiency of each step means only polypeptides <50 residues can be synthesized with high purity and yield Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 20 Chemical Synthesis of Peptides Fmoc • Protecting group for N-terminus • Removed before coupling DCC • Activating group for C-terminus of incoming amino acid Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 21 Chemical Synthesis of Peptides Lecture 13 Fall 2016 Biochemistry I Prof. Bellizzi 22 Circulardichroismspectroscopy • CD measures the molar absorption difference of left- and right-circularly polarized light • Chromophores in a chiral environment produce characteristic signals • CD signals from peptide bonds depend on the chain conformation • Diagnostic of whether protein is properly folded, what secondary structural elements are present. Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 23 Intrinsically Disordered Proteins Some proteins (up to 1/3 of human proteins) contain segments with flexible, disordered structures • Flexible linkers/spacers between domains • Involved in protein-protein or protein-DNA interactions (Can conform or mold to multiple different binding partners) Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 24 Folding Accessory Proteins • Protein disulfide isomerase (PDI) – catalyzes disulfide bond shuffling • Peptide prolyl cis-trans isomerase (PPI) incterconverts trans/cis Pro • Molecular chaperones • Isolate unfolded/misfolded proteins to prevent them from forming aggregates and give them time to fold properly • Two categories – heat shock proteins, chaperonins Heat Shock Proteins are produced in response to elevated temperature and bind and release unfolded or partially folded proteins to prevent aggregation In E.coli, the heat shock proteins are known as DnaJ and DnaK (Hsp70/Hsp90 in eukaryotes) Lecture 12 Fall 2016 Biochemistry I Prof. Bellizzi 25 Folding Accessory Proteins Chaperonins are present during normal conditions and facilitate proper protein folding • In E.coli, the main chaperonin is GroEL/GroES (Hsp60 in eukaryotes) • GroEL: Dimer of heptamers - tetradecamer(two back-to-back chambers) • GroES: heptameric cap • ATP hydrolysis causes conformational changes, gives protein time to fold
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