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Biochem ch. 3

by: Nadezhda L Zakhariya

Biochem ch. 3 CH 490 - 001

Nadezhda L Zakhariya
GPA 3.68

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About this Document

Goes over amino acids, different ways of analyzing peptide sequences, proteins and enzymes. Includes different chromatography techniques
Steve L. Reichow
Class Notes
biochemistry, Lehninger Principles of Biochemistry, Biochemistry 1
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This 13 page Class Notes was uploaded by Nadezhda L Zakhariya on Thursday October 13, 2016. The Class Notes belongs to CH 490 - 001 at Portland State University taught by Steve L. Reichow in Summer 2016. Since its upload, it has received 14 views. For similar materials see BIOCHEM STRUCTURE & FUNCTION in Science at Portland State University.


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Date Created: 10/13/16
03.10.2016 Lecture Notes - ​ Ch. 3 - Amino Acids, Peptides and Proteins Proteins ​: Main component in biological function by providing a means for… - Enzymatic Catalysis (​enolase , DNA polymerase) - Transport ​(hemoglobin → O​ transport, Lactose Permease → lactose transport) 2​ - Structure ​(connesctive tissue (Collagen),Hair, nails (Keratin)) - Motion ​(Myosin, Actin → both found in muscle tissue) Amino Acids (AA): ​ Protein building blocks - Proteins are ​heteropolymers:​ Polymer derived from 2+ different types of monomer AA Properties: ● Can for stable polymers ● Has useful Acid/Base Properties ● Varied Physical properties ● Chemical functionality varied Biochemical Designation (aka: Naming): AA’s are named starting from ⍺-carbon (⍺), and go down R-group with greek letters (β,γ,δ,ε…) * Concerned only with this naming system ⍺-carbon is the Chiral center -always has 4 substituents, tetrahedral (requirements to be chiral center) -Carboxyl & amino group, ⍺-Hydrogen, a unique R-group - glycine exception (2 H) - L (left) & D (right) in determining stereochemistry * Proteins have only L- Amino acids - Can draw molecule in standard or fischer projection (showing stereochemistry). *Know how to draw in fischer projection upon being given an AA 20 Common L - AA​s divide into 5 groups ● Nonpolar, aliphatic (7) ○ Glycine, GLy, G ○ Alanine, Ala, A ○ Proline, Pro, P ○ Valine, Val, V ○ Leucine, Leu, L ○ Isoleucine, Ile, I ○ Methionine,Met, M ● Aromatic (3)​ A side chains absorb UV light @ 270-280 nm ○ Phenylalanine, Phe, F ○ Tyrosine, Tyr, Y ○ Tryptophan, Trp, W ● Polar, uncharged (5)​ 2 Cystines can form disulfide bonds in oxidative environment, broken apart in reducing environment. (redox chem). Bonding also depends on proximity. ○ Serine, Ser, S ○ Threonine, Thr, T ○ Cysteine, Cys, C ○ Asparagine, Asn, N ○ Glutamine, Gln, G ● Positively Charged (Basic) (3)​ ​pKa ~6 for histidine R-group ○ Lysine, Lys, K ○ Arginine, Arg, R ○ Histidine, His, H - (in charged group as it can be protonated/deprotonated on R group) ● Negatively Charged (Acidic) (2) ○ Aspartate, Asp, D ○ Glutamate, Glu, E “Uncommon” AA’s exist. - Most arise via​ ​Post-translational modifica​ hemical modification of AA conducted by enzyme, after protein was synthesized from the information translated by ribosome from the RNA. The result is a ​Modified prot​ hese Modifications are REVERSIBLE, and important for regulation/signaling (esp. Phosphorylation), and important in metabolic pathways. - Examples of modifications: Phosphorylation (Phosphate group added on), Methylation (methyl group added on..) Acetylation, Adenylation. - In Cell Signaling Network: Transmitting of signals via change of protein phosphorylation level. - In Epigenetics: Signals sent to histones (&DNA), turning gene expression on/off. Via methylation Also “Un-natural” (synthetic) AA’s - Used to investigate function of proteins, or make interesting chemical properties. - Used in Protein Engineering field to create various proteins w/new structure and function - Ionization of AA Acid pH​ - ​fully protonated COOH protonated, NH3+ protonated Neutral pH - ​​ witterion COO- deprotonated, NH3+ protonated Basic pH ​- ​fully deprotonated COO- deprotonated, NH3+ deprotonated Uncharged AA sides will have 2 pKa values pKa of ⍺-carboxyl group = pK1 pKa of ⍺-amino group = pK2 The Carboxyl group deprotonates first by the first equivalent of OH-, followed by the amino group with the second equivalence of OH-. REMEMBER, there are buffer effects happening when pH is within one unit of the pKa (1 and 2) -ex: glycine → pKa1 - 2.34 pKa 2 - 9.6 → lower pKa = stronger acid Perfect zwitter ion occurs at the IP point. PI = (pKa1 + pKa2)/2 PH = PI → net charge is zero - least soluble point in water - Bad electric conductor What affects pKa? chemical environment.​ The ⍺-carboxyl group is much more acidic than carboxylic acids (stabilized by NH3+) and the ⍺-amino group is slightly less basic than an amine group (due to the withdrawing effect of the COO-) AA’s with ionizable chains → 3 pka’s​ (COOH, NH3+, R-group) - ex: Lysine, Histidine, Arginine.. Titration curves are complex and​ pKas hard discern unless more than 2 pH units apart How to calculate PI of AA with ionizable R-group? 1. Identify zwitterion (where AA overall neutral) 2. Find pKa that defines acid strength for this zwitterion (first pKa below it) 3. Find pKa that defines basic strength for this zwitterion (first pKa above it) 4. Take avg. of these two pKas *know how to draw titration curves *​(example problem in lecture notes). Properties of diff. AA’s define structure and function of peptides and proteins. Peptide Formation Formed between 2 AA’s via ​condensation reaction. ​ The reaction is ENDERGONIC, need energy→ biologically catalyzed by ribosomes, In cells the -OH is activated via ATP -->product of condensation = peptides. - Peptides small compared to proteins - Peptides are <10 kDa, ~100 amino acids or less ----Daltons (Da) Dalton equivalent to amu - 1Da = mass 1 proton Molecular mass in Da same as molar mass in g/mol Typically used in Da or kDa (1kDa = 1,000 Da) ---- Numbering and naming AA in chain starts from N to C terminus. Two ways of expressing: - Ex: Ser​ -G1​​ -Ty2​ -Al3​ -Leu4​ 5 - Ex: SGYAL IN LAB: Peptide bonding can be synthesized via F ​ moc​ (Protecting group) & ​DCC ​(activating grp.) - Solid-State Fmoc Chem. → most common - Solution-state is too difficult to purify - Peptides >100 AA are not practical. Yield drops. - Ribosome can synthesize 100AA in 5 seconds (1/10,000 error) :O woah! 1. Fmoc attached to N terminal of AA​ , attached to be1​ structure at C terminal. 2. Fmoc is removed via mild acid 2.5 Fmoc is attached to AA​ at N termi2 ​, and DCC is attached at C term. To activate it 3. AA​ +bead complex bonds with AA​ at N terminal of AA​ (Activated C terminal of AA​ ) 1​ 2 ​ 1​ 2​ 4. Fmoc is removed via mild acid *Steps 2.5 - 4 can be repeated over and over, placing more amino acids on the chain* ​ 5. Remove peptide from bead via strong acid (TFA) Relevance and Application Natural functions: Human adopted: Hormones & Pheromones As Pharmacological agents (drugs) - Ex: Insulin, Oxytosin, sex-peptide -mimics/competes with native protein interactions Neuropeptides As Biochemical tools - Ex: substance P (pain mediator) -Studying proteins in peptide fragments Antibiotics → often easier and cheaper - Ex: Polymyxin B, bicitracin *** Important to validate findings in context of a full protein*** Protection (toxins) - Ex: Apamin, conotoxin, chlorotoxin Proteins Peptides (<10kDa) Vs. Proteins (typically >10kDa) Talking about proteins, will mention: Polypeptides (Covalently linked ⍺-AA) ​ ​ ​ ​ ​ ​ ​ ​ -​Density​ based separation Machine spins given substance. Higher the speed of rotation (g) and the time spun for = smaller particles obtained -Precipitation​ ​(followed by centrifugation/filtration) -​Solubility​ based separation based on chem and thermal environment. -Chromatography -​Interaction with solid-phase matrix​ based separation Column chromatography:​ has mobile (protein solution) and stationary (porous matrix) phase - Proteins in mobile phase interact with the unique matrix of the stationary phase. Ion-Exchange Chromatography -​charge​ based separation Cation exchange: Resin negatively charged, positively charged proteins stick to it Anion exchange: Resin positively charged, negatively charged proteins stick to it Strongly bound proteins then eluted with high salt buffer Cation exchange or anion exchanged depending on the protein's PI and the buffer that it is in. - decide if the protein is positive (buffer pH<PI) or negative (buffer pH>PI) - Positive proteins → cation exchange Negative Proteins → anion exchange Size-exclusion chromatography ​(aka gel-filtration) - this technique can also be used to determine Mw of your protein Atrix/resin beads are porous allowing smaller proteins to travel through these canals, moving very slowly Larger proteins don't fit into channels and thus move faster past the beads Beads come in many pore sizes You can determine the bead needed by the its molecular weight in daltons -​ex: #AA in protein * 110 Da = _____Da Compare the size of protein to that of which the bead was made for, and choose appropriate bead. Affinity Chromatography -affinity​ to ligand based separation Bead tagged with a ligand (small molecule,metal,antibody/protein…) Protein will bind to the ligand on bead This ​method can be very specific ​as affinity “tags” can be genetically engineered Assessing quality of peptide/protein Purity of final product needs to be assessed For enzymes​: determine catalytic activity present (via enzymatic analysis) - Enzymatic/catalytic activity: ​amount of product formed per unit of time Enzymatic Analysis -Total Activity Measured in units. 1 unit = transforms 1umol product per min @ 25C How many units a sample has (irres-pective of surroundings) -Specific activity =​total Activity/Total proteins *​specific activity goes up as purification progresses For proteins: -​Gel Electrophoresis- - electric field separates proteins in gel matrix according to charge and shape/size Mobility is hindered by size of proteins (smaller → move faster) This is the basis for the following techniques.. Polyacrylamide Gel Electrophoresis (PAGE) - ​ charge & size/shape separation Denaturing Electrophoresis (SDS-PAGE) -​ just size separation SDS​ (a detergent) micelles bind and denatures proteins - all proteins become linear, and negative - Will separate by size, smaller proteins traveling faster SDS does not break disulfide bonds: B ​ ME or DTT​ incorporated to do so. Isoelectric Focusing - separate by PI and size of protein - Does not denature An electric field is ran through a gel strip with pH gradient -@ pH = PI the protein does not migrate Isoelectric Focusing + SDS-PAGE -combining the two Primary Sequence determination Primary sequence provides: - Protein ID - Physical-chem properties - Prediction of 2,3,4th structures - Insight into biological function, disease and evolutionary history 3 ways to determine: Determine from DNA sequence Look at DNA sequence, each codon stands for one AA. Very quick and cheap. Edman Degradation (classical method) Successive rounds of N-terminal modification, cleave, and identifying YAY​: requires 10-100 picomoles of protein NAY​: sequences up only 30 AA at N-terminus - can cut protein into smaller pieces first via: - Trypsin → cleaves Lys, Arg (@ C terminal) - Chymotropsin → Phe, Trp, Tyr (@ C terminus) - Cyanogen bromide (BrCN) → Met (C) - Overall: good for identifying proteins with known sequences Mass spectrometry (modern method) Electrospray Ionization Mass Spectrometry (ESI-MS) - protein placed in a high voltage field and gets sprayed into mass spectrometry - Proteins do not get cleaved YAY​: provides accurate mass measurements NAY​: no sequence info ESI Tandem Mass Spec. (ESI-MS/MS) In short: make fragments, get mass of fragments, organizes them Step 1: proteolysis (cut into peptides) Step 2: ESI-MS/MS ( cleaves peptide bonds) - organizes data in peaks of ions, differing by one AA from N-C terminus Primary Sequence Comparison Analysis Comparing sequences b ​ etween proteins, individuals and species Homologs​: Proteins gave similar sequences and functions (at least 30% identical) Orthologs:​ Homologs from different species Paralogs:​ Homologs from same species Can be used to find signature sequences that give distinguishing features by looking at the conserved part that is shared Sequence variations may be conservative or nonconservative (in size of AA as well) Practice getting those amino acids ;)


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