L-4 & 5 Noiva August 5 & 7, 1997
BIOC520: Medical Biochemistry PROTEIN STRUCTURE AND PROTEIN FOLDING
OBJECTIVES: 1. To understand the chemical properties of the peptide bond. 2. To learn the chemical forces that govern protein structure 3. To understand the 4 levels of protein structure 4. To understand the biological process of protein folding. 5. To understand sequencing of polypeptides 6. To understand gel electrophoresis of proteins READING: Devlin, Chapter 2, pp. 39-81. Marks, Chapter 8, pp. 79-98. CLINICAL CORRELATIONS: Ann Sulin - Insulin-dependent diabetes (Marks pp. 80, 88-89, 97) LECTURE: I. Peptide bond - amide bond between alpha-amino and alpha-carboxyl groups of 2 amino acids A. Chemical properties (Fig. 2.9 Devlin: cis, trans peptide bond) 1. Peptide bond is polar and planar (Devlin Fig. 2.10, pg 29, Marks Fig 8.5, pg 81) a. electron resonance structure (Devlin Fig. 2.9, pg 28) b. has partial ( 40%) double bond character c. amide group is planar, usually trans (Marks Fig 8.6, pg 82) 2. Synthesis (Devlin Fig. 2.8, pg 28) a. condensation produces water b. energy required (ATP hydrolysis) 3. Peptide bond is hydrolyzable a. acid hydrolysis generates free amino acids i. 6N Hydrochloric acid heated at 110øC for 24 hr in a vacuum b. base hydrolysis generates free amino acids i. 4N Sodium hydroxide heated at 100øC for 4 hr c. cyanogen bromide cleaves at the COOH-terminal side of Met d. enzymatic hydrolysis of peptide bonds by proteases i. peptidases are specific for certain amino acids (Devlin Fig.2.65, pg 75) Learn the specificity of the proteases. 4. Polypeptides are polyampholytes a. ampholyte has both acidic and basic pKa values b. isoelectric point - pH at which the net charge is zero For example: pKa of the Alpha-carboxyl group = 3.6 H3N+-Ala-Lys-Ala-Ala-COO- pKa of the Alpha-amino group = 8.0 pKa of the delta-amino of the Lysine = 10.6 at pH = 1 the net charge is +2 at pH = 6 the net charge is +1 at pH = 14 the net charge is -1 the isoelectric point pI = (pKa2 + pKa3)/2 = (8 +10.6)/2 = 9.3 B. Nomenclature 1. Size a. dipeptide, tripeptide, etc. b. oligopeptide - several amino acids (up to 20) c. polypeptides (more than 20 amino acids) - all proteins are polypeptides II. Physical Forces Governing Protein Conformation A. Physical forces govern 3-D structure of proteins (Pauling and Corey) 1. bond lengths and angles should be distorted as little as possible 2. structures must follow Van der Waal's rules for atomic radii 3. peptide bond is planar and trans 4. noncovalent bonding stabilizes structure 5. conformation can change without breaking bonds (flexibility) B. Types of noncovalent forces important to protein conformation 1. Hydrophobic forces a. hydrophobic residues orient to inside b. hydrophilic residues orient out 2. Van der Waal's potential (Devlin Fig. 2.51, pg 67) - includes electron shell repulsion, dispersion forces, and electrostatic interactions 3. Salt bridges, electrostatic forces 4. Hydrogen bonds (Devlin Fig. 2.48, pg 66) C. Angles of rotation of the polypeptide chain determine structure (Devlin Fig. 2.24, pg. 43) 1. angles of rotation around alpha-carbon are (psi and phi) a. (psi) is the angle of the alpha-carbon bond to the carbonyl-carbon b. (phi) is the angle of the alpha-carbon bond to the amide-nitrogen 2. primary sequence and angles of rotation for each alpha-carbon completely define protein conformation 3. only a small number of psi and phí angles are allowed 4. statistical analysis of all proteins yields goups of prefered angles a. areas of repeating (psi) and (phi) angles are secondary structures III. Levels of Protein Structure A. Primary Structure - amino acid sequence of a polypeptide 1. primary structure determines 3-dimensional structure (Anfinsen) 2. always represented NH2-terminus to COOH-terminus B. Secondary structure - regular local conformation of linear segments of the polypeptide chain 1. Secondary structure are stabilized by hydrogen bonds between amide and carbonyl groups 2. Several types of secondary structure a. alpha-helix (Devlin Fig. 2.26, pg 44) 1. right handed helix 2. 3.6 amino acids per turn 3. carbonyl oxygen hydrogen bonded to 4th amide hydrogen 4. amino acid R-groups orient out 5. proline breaks the helix b. beta-pleated sheet (Devlin Fig. 2.27-2.29, pg 45) i. polypeptide chains side by side ii. polypeptide chains can be parallel or antiparallel iii. carbonyl oxygen hydrogen bonded to amide hydrogen iv. beta-strand is a single pass of the polypeptide c. reverse turn, beta-bend (Marks Fig. 8.10, pg 85) i. allows a sharp turn in polypeptide chain ii. carbonyl oxygen hydrogen bonded to 4th amide hydrogen iii. Glycine is required 3. Fibrous proteins demonstrate secondary structure a. Fibroin i. silk is fibroin ii. antiparallel-beta-pleated sheet b. alpha-Keratin and tropomyosin i. alpha-keratins in wool and hair and epidermal layer ii. tropomyosin is a thin filament in muscle iii. alpha-helix allows elasticity iv. alpha-keratin converts to beta-pleated sheet with heat or stretching - disulfides are important to maintenance of keratin secondary structure - alpha-keratins are beta-pleated sheets; feathers and claws c. Collagen (Devlin Fig. 2.38, pg 53) i. structural protein; skin, bones ii. triple helix (not alpha-helix) iii. sequence (Gly-Xaa-Pro)x or (Gly-Xaa-HyPro)x iv. glycine required for triple helix to form v. contains many modified amino acids vi. hydroxyproline stabilizes the structure vii. vitamin C required for hydroxylation; scurvy C. Tertiary structure - overall folded conformation of the polypeptide 1. Physical forces affect tertiary structure a. Hydrophobic forces i. hydrophobic residues orient to inside ii. hydrophilic orient out b. salt bridges, electrostatic forces c. Van der Waals radii d. Hydrogen bonds e. Disulfide bridges D. Quaternary structure - subunit structure 1. aggregation of 2 or more subunits a. hetero- or homo- polymers 2. same forces drive tertiary and quaternary structure E. Structural elements 1. Sequence motif - small functional linear polypeptide sequence (may not be 3-D) a. signal peptide b. ER-retention signal c. mitochondrial and nuclear targeting signals d. RGD cell adhesion motif 2. Supersecondary structure (structural motif) a. smallest conformational unit (may be functional) b. examples (Marks Fig. 8.11, pg 85) - àà (helix-loop-helix), à , -hairpin - beta-barrels (Devlin Fig. 2.34, pg 48) - Rossman fold, a nucleotide binding site - leucine zipper mediates transcription factor dimerization - zinc finger is a DNA binding motif 3. Domain (Devlin Fig. 2.36, pg 49) a. the part of a polypeptide chain that can independently fold into a tertiary structure b. often domains have units of function c. proteins may contain one or many domains IV. Protein folding A. Folding occurs step-wise with several intermediates (Fig. 8-5, 7) unfolded/secondary structure/domains/molten globule/native tertiary structure 1. a collapsed structure (molten globule) occurs very quickly 2. steps between molten globule and native tertiary structure usually occur slowly a. intermediates are isolatable b. multiple pathways are possible B. Folding is driven by hydrophobic forces C. Proteins can self assemble but in vivo folding is facilitated by proteins 1. Chaperones are binding proteins which assist folding (Devlin Fig. 2.46, pg 64) a. chaperones cause misfolded protein to unfold rather than aggregate b. many chaperones require ATP hydrolysis for activity c. more than one chaperone may act simultaneously and sequentially in the folding of a single protein d. chaperones are specific for specific protein synthesis pathways (cytosolic vs. mito. vs. endoplasmic reticulum) 2. Enzymes catalyze kinetically slow steps infolding a. cis-trans prolyl isomerase i. both cis and trans peptide bonds to proline naturally occur ii. the isomerization of peptidyl-proline bonds may be slow b. protein disulfide isomerase i. catalyzes disulfide bond formation and isomerization D. Denaturation is unfolding 1. Requires some input to overcome hydrophobic forces a. heat b. denaturant (urea or guanidinium) 2. Requires reductant to reduce disulfide bridges to sulfhydryls V. Analytical Techniques in Protein Biochemistry A. Determination of Amino Acid Composition 1. Amino acid analysis provides % of each amino acid in protein a. Hydrolysis of polypeptide with 6N HCl b. Derivitization of amino acids with dansyl chloride PITC, or 0-phthalaldehyde (OPA) c. Liquid chromatographic separation of the tagged amino acids d. Quantitation 2. Composition of a protein can be used to identify a protein B. Determination of primary sequence of a polypeptide 1. Preparation of peptides for sequencing a. Removal of disulfide bridges i. reducing agent ( -mercaptoethanol or dithiothreitol) ii. derivatize sulfhydryls to block disulfides from reoxidizing b. Digestion with cyanogen bromide i. CNBr cleaves at the carboxyl side of methionine residues c. Digestion with proteolytic enzymes i. use at least two different enzymes ii. overlapping enzymes allows determination of peptide sequence (Devlin Fig. 2.65, pg 75) d. Separation of peptides i. peptides separated by chromatography ii. based on differences in ionic, polar, and/or hydrophobic characteristics 2. Edman degradation is used to sequentially determine a.a. sequence (Devlin Fig. 2.64, pg 75) a. PITC reacts with the N-terminal amino acid b. Strong acid cleaves the peptide bond between the 1st and 2nd amino acids c. Product is a PTH derivative of amino acid #1 d. Determine identity of amino acid-PTH using HPLC chromatography e. Repeat steps a-d Note: Edman degradation has limited success with very long polypeptides C. Determination of Molecular Mass 1. Gel Filtration (Molecular exclusion chromatography; Devlin Fig. 2.61, pg 72) a. protein is loaded on a column of porous beads b. small molecules can enter the beads, large ones cannot c. an aqueous buffer moves the protein through the beads (Figure of SDS-Polyacrylamide Gel Electrophoresis setup) 2. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) a. protein is unfolded and coated with sodium dodecyl sulfate (SDS) detergent b. proteins are loaded on an acrylamide gel matrix c. electricity moves the proteins through the matrix d. low molecular weight proteins move faster (farther) d. large molecules migrate faster because they bypass beads D. X-ray Crystallography and NMR 1. Physical techniques to identify 3-D structure of a pure protein 2. Requires tremendous time, effort, and analytical resources
Go to Previous BIOC520 Lecture
Go to Next BIOC520 Lecture
Go to Listing of Lectures
Go to BIOC520 Home Page
jt 9/96