Amino Acids Pratt & Cornely Chapter 4 Alpha carbon Sidechain Proteins peptides Amino Acid Structure 1
L amino acids Glycine R/S vs D/L L isoleucine racemization Stereochemisty Common Amino Acids 2
Which amino acid(s)... Is achiral? as a secondary amino group? as a chiral sidechain? Form these derivatives: Charged amino acids ther amino acids are ionizable Acid/base chemistry 3
Ionization of Amino Acids Polyprotic acids Alanine pka 1 = 2.4 pka 2 = 9.9 Zwitterion Isoelectric point More acidic than typical carboxylic acid Titration Curve 4
Titration Curve Which amino acid? Ionization States f Amino Acids G E F A B C D 5
Disulfide bond formation Amide bond Primary structure and C terminus Condensation and hydrolysis Peptide bonds 6
Polypeptides Drawing Peptides Sidechains Stereochemistry Ionization states Example: Draw the peptide ASCVE at p 8. Steps Backbone Stereochemistry Sidechains Check ionization 7
Example: Draw the peptide ASCVE at p 8. Step 1 3 Step 2 3 Step 3 C 3 pka 2 3 pka 9 Step 4 pka 6 S pka 8.4 pka 4.1 C 3 3 pka 6 S but significantly deprotonated pka 4 8
Basis of secondary Structure Polarity Rigidity Cis/trans Conformational Constraint T cis/trans 9
Dihedral Angles Ramachandran Plots 10
Ramachandran Plots Alpha elix Right handed Polarity n and n + 4 Gly and Pro 11
elical Wheel Problem 31. The sequence of a domain of the gp160 protein(iv) is shown below using one letter codes for the amino acids. Plot this sequence on the helical wheel. What do you notice about the amino acid residues on either side of the wheel? MRVKEKYQLWRWGWRWG elical Wheel The sequence of a domain of the gp160 protein(iv) is shown below using one letter codes for the amino acids. Plot this sequence on the helical wheel. What do you notice about the amino acid residues on either side of the wheel? MRVKEKYQLWRWGWRWG W K W G Y G Q M R E K R R W V L W 12
Beta Sheets Alternating sidechains can lead to amphipathic sheets 13
Irregular Secondary Structure onrepeating loops and turns Change of direction Turns have about 4 residues Internal bonds Gly, Pro Tertiary Structure Too many shapes to memorize But not an infinite number of possibilities Take away the ability to read a paper Discussions of motifs and why important Discussion of domains and why important 14
Motifs (Super Secondary Structure) Recognizable combinations of helices, loops, and sheets Match elix loophelix elix bundle airpin sandwich Some Motifs are highly studied Know the lingo Leucine zipper Zinc finger ften have recurring applications Studying Motifs 15
Discrete, independently folded unit (may maintain shape when cleaved on loop) May have separate activities: ATP binding domain or catalytic domain Similar activity = similar structure across many proteins Binding pockets at interfaces Domains ow many domains? Common Domains 16
Major classes of globular proteins Mostly Mostly / combination Little secondary Thermodynamics of protein folding G might be 40 kj/mol for small protein (about 2 bonds) ydrophobic effect is important...but the most important? 17
Thermodynamic Contributions to Rase Pace, C.. Meth. Enz. 1995, 259, 538 554. Ion pair (salt bridge) Zinc finger Disulfide bond ther Stabilization 18
Protein Folding Denature/renature ative structure Domains fold separately Global vs. local minima Molecular chaperones Problem 40. Proteins can be denatured by agents that alter the balance of weak noncovalent forces that maintain the native conformation. ow would these agents cause a protein to denature? eat, p, detergent, 2 mercaptoethanol Protein processing 19
Thermodynamics and Kinetics Levinthal s paradox: not random sampling of all possible conformations Energy funnel Series of irreversible steps Entropy traps Quaternary Structure Multiple subunits: ligomers omodimer, heterotrimer Advantages Economy Stability regulation 2 3 2 2 20
Chromatography Protein Purification Affinity PLC Size Exclusion (gel filtration) Ion exchange Electrophoresis Isoelectric focusing SDS PAGE Size exclusion Chromatography 21
Ion Exchange Chromatography Isoelectric Focusing From wikipedia 22
Sodium dodecyl sulfate Polyacrylamide gel electrophoresis Analaysis technique Small proteins move faster SDS PAGE Amino Acid Sequencing Purify protein Break disulfide bonds Enzymatic digest (protease) Edman degradation verlap fragments 23
Partial Digestion Partial Digestion 24
Edman Degradation Sequencing ligopeptides 25
X Ray crystallography 26