Amino Acids and Proteins Notes, Lecture notes of Biochemistry

Download Amino Acids and Proteins Notes and more Lecture notes Biochemistry in PDF only on Docsity! Chem 145: Biochemistry I Notes I. Amino Acids 1. Unpolar (Hydrophobic) Amino Acids Proline P Leucine L Alanine A Valine V Tryptophan W Isoleucine I Methionine M Phenylalanine F 2. Polar (Hydrophilic) Amino Acids Glutamine Q Asparagine N Glycine G Serine S [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ Cysteine C Threonine T Tyrosine Y 3. Polar (Hydrophilic) Charged Acidic pKa (R) Aspartic Acid D ~4.0 Glutamic Acid E ~4.0 Basic Histidine H ~6.0 Lysine K ~12.0 Arginine R ~12.0 pKa (Cα) = ~2.0 pKa (Nα) = ~9.0 Essential Amino Acids PVT TIM HALL -Phenylalanine -Valine -Threonine -Tryptophan -Isoleucine -Methionine -Histidine -Alanine -Leucine -Lysine As a human grows into adulthood, the number of essential AA’s become 8 [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ Note: Tyrosine is not essential because: P[O ] ↔ Y examples: 1. Asp, Ser, Lys Separation based on charge: Asp = -1 Ser = 0 Lys = +1 D elutes first on a cation-exchange column, then S, then K 2. A, G Sep’n based on IMFA A(R=CH3)= ion-induced G(R=H2)= ion-dipole A elutes first because G has stronger IMF formed with the column 3. A,V Sep’n based on size Alanine is smaller = more polar, Valine elutes first II. Proteins -polymers of amino acids -read from N-terminal to C-terminal -peptide bonds formed from alpha-C of amino acid and alpha-N of the other amino acid Residue = amino acid in a protein “Oligo” – 2-20 residues “Poly” - >20 residues The Peptide Bond -usually found in trans- configuration -has partial (40%) double bond character -about 0.133 nm long Note: trans- configuration allows side chain (R) separation; no steric strain (n-1) = peptide bonds Where: n= number of residues Peptide planes In one peptide plane, the degree of freedom is 3 since there are 3 single bonds that are free to rotate n=5 # of peptide planes = n-1 = 4 # of peptide bonds = n-1 = 4 Peptide planes contain the peptide bonds Length: - > = > ≡ Partial double bonds are longer than double bonds C=O and C=N; N is bigger than O so C=O<C=N Monomeric – one polypeptide chain Homomultimer – 1 kind Heteromultimer – more than 1 kind eg. Hemoglobin (Hb) – tetramer heterotetramer – 2 alpha and 2 beta homomultimers (2 dimers) Size: Proteins are classified according to shape, solubility, and function (1) Shape – globular and fibrous [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ Globular – more diverse functions, generally soluble Fibrous – structural functions, long, rod-like shape, generally more stable Levels of Structure: 1. Primary (10) – final structure that the protein adapt, sequence held together by peptide bonds 2. Secondary (20) – IMFA is the major driving force in structure - local folding (H-bond is formed between carbonyl oxygen and amino hydrogen) -majorly stabilized by H-bonds -directionality: C=O to N-H -α−helices∧β−sheets 3. Tertiary (30) – folding of secondary structure -overall 3D shape -IMFA driving force -all favorable IMF’s in order to form: compact, folded, stable structure -allow forms for intermolecular S-S bridges 4. Quaternary (40) – subunit organization -no quaternary structures for monomeric proteins -can form S-S bridges which are either inter- or intra-molecular -can for intra- and inter-molecular H-bonds eg. Collagen -fibrous -made up of tropocollagen – triple helix as a secondary structure Myoglobin (Mb) – storage protein Hb – transport protein Psi and Phi angles [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ Steric combinations on psi and phi Phi = 0o, Psi= 180o Phi = 180o, Psi=0o Phi= 0o, Psi= 0o Helix Handedness eg. Tropocollagen – 3 left-handed helices coiled to form a right-handed triple helix Classes of Secondary Structures: - Alpha helix - Other helices - Beta sheets (compound of beta strands) - Tight turns (beta turns/beta bends) - Beta bulge Beta sheet – loose alpha helix Note: side chains are protruding outwards of a helix (generally hydrophilic interaction with solvent) Interaction among side chains may affect stability. H-bonds must form in order to form the structure. We need at least 8 residues to form an alpha-helix. Amino Acid helix breakers GLYCINE AND PROLINE -destabilize the helix Gly – too flexible due to its small nature Pro – rigidity due to the ring Alpha helix – intramolecular H-bonding Beta sheet – intermolecular H-bonding [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ – Contain the catalytic site (enzymes) – DNA-binding (in transcription factors) – Providing a surface to bind specifically to another protein Tertiary Structures – compact, stable -only multimeric proteins can form a quaternary structure Disulfide bond formation C+C [O ] ⇔ Cystine Protein Folding -dictated by primary structure 1. Slow Step – adaption of several conformations until its gets to a structure near its final structure 2. Fast Step Molecular Chaperones -help molecules to fold properly -increase the rate of correct folding of nascent Predictive Algorithms for Protein Structure -Chou-Fasman – based on relative frequencies of occurrences of the different AA residues Quaternary Structure – for multimeric proteins -adapt a symmetric pattern for minimal energy consumption -proteins can have more than 2 subunits (subunits – individual peptides) Isolation and Purification of Proteins -no one technique -at the isoelectric point, IMFs that will be formed are weaker than when charged (ion- dipole) -Gel filtration is the same as gel permeation, size exclusion, and molecular sieve Salting in and Solubility Salting out – if the concentration of the salt is > 0.1 M, the proteins salt out of the solution, increasing the ionic strength and decreasing the solubility of the protein in the solvent -this is because the salt is more mobile than the side chain of the protein in the solution and it competes with the side chain for ion- dipole interactions with the solvent -the decrease in solvation and neutralization of the repulsive forces allows the protein to aggregate and precipitate [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ Protein Techniques 1. Dialysis and Ultracentrifugation Dialysis -a solution of protein is separated from a bathing solution by a semipermeable membrane -useful for removing small molecules from macromolecular solutions or for altering the composition of the protein-containing solution Ultracentrifugation -improvement of the dialysis principle -filters with pore sizes over the range of biomolecular dimensions are used to filter solutions to select for molecules in a particular size range -requires high pressures because sizes are microscopic -useful for concentrating dilute solutions of macromolecules 2. Ion-Exchange Chromatography -the salt solution (counterion) used to wash the column is added in increasing concentrations to elute the different proteins 3. Size-exclusion Chromatography -if a molecule is too large to enter the pores on the beads of the column, it is excluded from the column at an elution volume -the smaller the volume, the greater the elution volume 4. Electrophoresis -based on the movement of ions in an electrical field -generally, electrophoresis is carried out not in free solution but in a support matrix such as polyacrylamide or agarose, which retards the movement of molecules according to their dimensions relative to the size of the pores in the matrix -all proteins with low isoelectric points will be more attracted to the anode, thus will be found nearer the anode Analysis of Proteins N-terminal Analysis -Edman degradation – allows sequential identification -Edman reagent – phenylisothiocyanate C-terminal Analysis -enzymatic approach Carboxypeptidases – cleave from C-termini sequentially Types: 1. A – any except Pro, Arg, Lys 2. B – Arg, Lys 3. C – any 4. Y – any Fragmentation of Polypeptide Chains -usually enzymatic; can be done by specific or nonspecific chemical means 1. Trypsin – Arg, Lys 2. Chymotrypsin – Trp, Tyr, Phe 3. Clostripain – Arg [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/ 4. Lys-C – Lys 5. Staphylococcal protease – D, E 6. CNBr – Met → sulfonium → cyclic iminolactone -other chemical means 7. Hydroxylamine (NH2OH) a. at pH 9 – Asn-Gly b. at pH 2.5 – Asp-Pro [1] - https://en.wikipedia.org/wiki/Schiff_base#Biochemistry [2] - https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and- domains/