Amino Acids Structural Features. Chapter 3 Amino Acids, Peptides and Proteins. Amino Acids Structural Features

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1 Chapter 3, Peptides and Proteins Amino acids can be formed from inorganic compounds under prebiotic conditions These compounds are the monomeric units that are joined to form polypeptides (aka. Proteins) Chapter 3 2 Structural Features All proteins are built from a combination of the 20 amino acids C α p R C COO R C COO - Carboxyl Amino N 2 + N 3 Amino acids are built around a central α-carbon atom Because an acid and a base are both present, an amino acid can form either a positive or negative ion In fact, at physiological p, both the acid and the base are completely ionized, resulting in a compound with both positive and negative charge called a Zwitterion Chapter 3 3 Structural Features Four different substituents on the α-carbon atom means that the alpha carbon is a chiral carbon (Enantiomers!!) These chiral centers are optically active, meaning they rotate plane-polarized light Rotating light to the left Levorotatory (L) Rotating light to the right Dextrorotatory (D) To determine whether your amino acid (or sugar!) is D or L, you need to look at the fischer diagram with the carboxyl at the top and the R group on the bottom. If the amino group is to the LEFT then the amino acid is L! Chapter 3 4 1

2 Structural Features Asymmetry (D vs. L) and side chain differences result in great variety in the polypeptides formed The amino acid residues in proteins are exclusively L stereoisomers. There are other L-amino acids in living cells Some as biochemical intermediates Some with modified R-groups after synthesis D-amino acids have been found in only a small number of peptides: The peptides of bacterial cell walls and in antibiotic peptides. Overall, ~ 300 different amino acids occur in living organisms!! ives nature a lot to work with when putting together proteins! Chapter 3 5 Classification of All amino acids have the same core structure, but vary in their side chains (the R group), which effects their physiochemical properties R C COO - N 3 + The variation in the R group allows the amino acids to be grouped based on the Polarity of their R group They fall into five distinct groupings as clusters of 7, 5, 3, 3, and 2: the aliphatics, polars, aromatics, basics, and acidics ydrophobic Water fearing Non-polar side chains ydrophilic Water Loving Polar, neutral or charged side chains Chapter 3 6 The Seven Aliphatics (Non-polar) The Five Uncharged (Polar) These seven amino acids tend to cluster together within proteins, stabilizing structure through hydrophobic interactions. The R groups of these five amino acids are more soluble in water than those of the alphatic AAs due to the presence of hydroxyl, thiol and amide groups. Chapter 3 7 Chapter 3 8 2

3 The Three Aromatics The Three Basics These three amino acids are relatively non-polar and all can participate in hydrophobic interactions. They all also absorb UV light due to their conjugation. The hydroxyl group of Tyr also allows this residue to form hydrogen bonds. Chapter 3 9 These three amino acids (along with the acidic AAs) are the most hydrophilic AAs and can participate in hydrogen bonding interactions as -bond Donors. Chapter 3 10 The Two Acidics These two amino acids (along with the basic AAs) are the most hydrophilic AAs and can participate in hydrogen bonding interactions as -bond acceptors. Other ways to roup the AAs Which and how many have hydroxyl groups? contain sulfur? contain 5- or 6-membered rings? have pure hydrocarbon side chains? have nitrogen in their side chains? Which is biggest? smallest? most acidic? most basic? Chapter 3 11 Chapter

4 Classification of Chapter 3 13 Chapter 3 14 Essential Vocabulary for CE 421 You will be required to learn the structure, name, abbreviation, pk a values (average ones for the carboxyl and aminol; specific value for ionizable R groups!) and unique characteristics of each of these 20 amino acids. You will need command of these structures to be successful in the rest of this course Unusual L- Found in Proteins In addition to the 20 standard AAs, proteins can (and do!) contain residues formed from the modification of common residues already incorporated into a polypeptide. Selenocysteine is a rare AA that is introduced into the protein as the modified version rather than modified once in place. Fig. 3-8a Sometimes referred to as the 21 st Amino Acid Chapter 3 15 Chapter

5 Unusual L- Found Elsewhere Not all AAs are found in polypeptides. Many are found in metabolic and synthetic pathways. In what synthetic (ornithine) and degradation (citrulline) pathways do you think that ornithine and citrulline show up as intermediates? Fig. 3-8a Chapter 3 17 The Amino Acid as Zwitterion An AA has at least one acidic (carboxyl) and one basic (amino) group If both are charged at the same time, the AA is a zwitterion The R-group may also contribute charge Y, C, K,, R, D and E Due to these multiple ionizable groups, a titration curve for an AA will have a minimum of two inflection points What happens to the structure above and below the pk a values for the amino and carboxyl groups? What form would be present at physiological p, 7.4? Chapter 3 18 Titration of an Amino Acid Titration of a Charged Amino Acid Where does the zwitterion form occur? If the pk a of the carboxyl group of acetic acid is 4.76, why is the pk 1 of glycine s carboxyl group so much lower, at 2.34? ow would the titration curve of istidine vary? Why is the pk 1 of istidine s carboxyl group 1.82 compared to 2.34 for lycine? ow would the titration curve of Lysine vary? Chapter 3 19 Chapter

6 Titration of an Amino Acid Local Chemistry Affects the Charge Properties of Isoelectric Point (pi) The characteristic p at which the net electric charge of the amino acid is zero is the isoelectric point (pi) For amino acids with a non-ionizable R group, this value is equal to the average of the two pk a values: pi = ½ (pk 1 + pk 2 ) pk 1 lies between p Chapter 3 pk 2 lies between p At p values above the pi what is the overall charge on the amino acid? What about below the pi? Why would this be good info to know? Chapter 3 22 Titration of a Charged Amino Acid What about the pi for AAs with an ionizable R group? Where s the Zwitterion? Chapter 3 23 Titration of a Charged Amino Acid Isoelectric Points for with Ionizable R-groups If an amino acid has two amino groups, or two carboxyl groups, the pi is equal to the average of the values of the two like groups. Example: L-istidine pk 1 = 1.82, pk 2 = 6.00, pk 3 = 9.17 Therefore: pi = ( )/2 = 7.59 Why does this rule of thumb work? Chapter

7 Can You Estimate the Number of in a Protein? The answer is yes! First, you need to know that the weighted average MW of the 20 standard amino acids is 128. Next, you take the molecular weight of the protein and divide it by 110 (WY? What happened to the other 18?) e.g. a protein of 55 kd = MW 55,000 is comprised of about 500 amino acids Chapter 3 25 Functional roups in Macromolecules Can Provide Buffering Capacity Chapter 3 26 But Enzyme Catalysis is Also p-sensitive Let s See Who Learned Their AAs this Weekend Why might this be? Think about it Amino Acid Quiz Chapter 3 27 Chapter

8 Peptides and Proteins Proteins Result from Peptide Bond Formation (a Condensation Reaction) Poor leaving group The peptide bond is formed through a nucleophilic displacement that takes place in the ribosome during translation. ood nucleophile = + 21 kj/mole (very unfavorable so why are proteins so stable?) Peptide bond Chapter 3 29 Peptides and Proteins Can You Estimate the Isoelectric Point of a Protein? Review the rule of thumb for calculating the pi s of amino acids. ow might this rule apply to peptides and proteins? Remember that polypeptides contain only one free α- COO and α-n 2 group, at opposite ends of the chain The R groups of some of the AAs in the polypeptide will be ionizable and contribute to the pi of the protein. Remember, the pk a values for both the termini and the R groups are effected by the chemical environment, so their use in this type of calculation will give you an estimated pi only (better to determine in the lab!) Chapter 3 30 Peptides and Proteins A Pentapeptide Peptides and Proteins AA Composition of Proteins Each protein has a characteristic mix of AAs in its sequence. No two proteins are identical in this makeup. Some AAs appear frequently and other not so much. What is the AA sequence? What is the approximate pi? Chapter 3 31 Chapter

9 Working with Proteins pi and AA Composition Amino Acid Ala Arg Asn Asp Cys ln Lysozyme # in PP (%) 12 (9.3%) 3 (2.3%) 11 (8.5%) 5 (3.9%) 8 (6.2%) 5 (3.9%) Pepsin A % in PP 16 (4.8%) 3 (0.9%) 15 (4.5%) 22 (6.7%) 6 (1.8%) 15 (4.5%) Peptides and Proteins Multimeric Proteins lu 2 (1.6%) 14 (4.2%) ly is Ile Leu Lys Met Phe Pro Ser 12 (9.3%) 0 (0.0%) 5 (3.9%) 9 (7.0%) 17 (13.2%) 1 (0.8%) 2 (1.6%) 2 (1.6%) 9 (7.0%) 34 (10.3%) 1 (0.3%) 30 (9.1%) 21 (6.4%) 0 (0.0%) 3 (0.9%) 15 (4.5%) 15 (4.5%) 49 (4.8%) Average AA molecular weight is 110. We can use this value to estimate the number of AAs in a protein if we know the MW of that protein. Thr 8 (6.2%) 26 (7.9%) Trp 5 (3.9%) 5 (1.5%) Tyr 5 (3.9%) 17 (5.2%) Val 8 (6.2%) 23 (7.0%) # of Negatively Charged Residues 7 36 # of Positively Charged Residues 20 3 Chapter 3 33 pi Multisubunit proteins consist of two or more separate polypeptide chains. If at least two of these chains are identical, the protein is oligomeric and each chain is a protomer Chapter 3 34 Peptides and Proteins Conjugated Proteins In addition to AAs, many proteins contain permenantly associated chemical groups called prosthetic groups These groups are often involved in the catalytic activity of enzymes or in structural maintainence Conjugated proteins are classified on the basis of the attached prosthetic group. Proteins can also contain more than one of these groups Chapter 3 35 CP 3.4 Peptides and Proteins Protein Structure Beyond the AA Sequence CP 4 Chapter

10 Working with Proteins Separation and Purification of Proteins A pure sample of protein is vital to determining its structure and function Proteins can be separated by exploiting their differences which are determined by their structure and amino acid sequence Solubility Ammonium Sulfate Precipitation Crystallization Charge Size Specific Binding Special Properties Ion Exchange Chromatography Isoelectric Focusing el Filtration Chromatography SDS-PAE Electrophoresis, Dialysis Affinity Chromatography eat Denaturation Engineered Properties Chapter 3 37 Working with Proteins Separation and Purification of Proteins Cell Lysis Liquid chromatography Centrifugation to remove cell debris/insoluble components Soluble Fraction Removal of nucleic acids (Optional) Protein Fraction Crude fractionation by precipitation Purified Enriched protein Liquid protein fraction Chapter 3 chromatography 38 Working with Proteins Column Chromatography In any type of column chromatography, you have the mobile phase and the stationary phase There are two types of stationary phases: Non-adsorptive: No interaction between the solute and the SP Adsorptive: Chemical interaction between the solute and the SP. You must alter your MP to elute the solute. As with all chromatography, you must equilibrate you column with MP prior to loading and eluting your solute! Working with Proteins Column Chromatography Ion Exchange (IEC) Proteins differ in the number of charged amino acids (Asp, lu, is, Arg, and Lys) found in their AA sequence. Therefore, they will vary in their overall charge at various p values and the p value at which their net charge is zero (their isoelectric point, pi) In IEC, the SP contains charged groups: Anion exchange (+++) / Cation exchange (- --) Strong and weak ion exchangers are available depending on your protein Sample eluted with increasing salt or change in p Isoelectric point of protein generally needs to be known or several pilot runs need to be performed Chapter 3 39 Chapter

11 Working with Proteins Column Chromatography Size Exclusion (SEC) A protein s size and shape affect its ability to move through a matrix of of porous beads or a porous membrane Proteins elute in decreasing order of size The SP is a non-absorptive & porous matrix SP with varying pore sizes are available Some uses include: Separating contaminating solutes and Protein fractionation Chapter 3 41 Working with Proteins Column Chromatography Affinity (Aff) Proteins can naturally or artificially contain special binding properties: Receptor-ligand interactions Protein-protein interactions Samples eluted by various means dependent on the binding interaction The SP contains specific ligands dependent on the binding property being exploited: Dyes Metals Substrates and cofactors Aff has a very high selectivity that often yields pure or nearly pure protein in minimal steps. Chapter 3 42 Working with Proteins Electrophoresis Electrophoresis is a method of molecule separation that exploits the size and charge of the components ere, the application of an electrical field to macromolecules in solution will cause them to migrate to either the: + anode (negatively charged molecules) cathode (positively charged molecules) els typically used as a support matrix act as molecular sieves through which molecules may or may not be able to pass based on their size: Agarose (DNA) and Polyacrylamide (Proteins) Chapter 3 43 Working with Proteins Electrophoresis SDS (sodium dodecyl sulfate) binds to proteins via hydrophobic interactions Usually about 1 SDS per 2 AAs When SDS added in excess: proteins coated with SDS, thus masking charge of protein and imparting overall negative charge proteins are denatured, meaning organizational structure of molecule is disrupted To completely denature protein, add a reducing agent such as β-mercaptoethanol (BME), dithiothreitol (DTT) and/or heat O Na + - O S O (C 2 ) 11 C 3 SDS-PAE O Sodium dodecyl sulfate (SDS) Chapter

12 Working with Proteins Electrophoresis Determination of MW Working with Proteins Isoelectric Focusing Procedure used to determine the pi of a protein ere, a p gradient is established by allowing a mixture of low MW organic acids and bases distribute themselves in an electric field generated across a gel. When a protein mixture is added, the protein components migrate until they reach a p that matches their pi value. Let s try Cytochrome c Peroxidase It contains 295 AA s Chapter 3 45 pi / Molecular Mass Calculator Chapter 3 46 Working with Proteins 2-D Electrophoresis 2-D electrophoresis combines SDS-PAE and IE into one experiment. This allows for the separation of proteins based on both MW and pi. Let s us separate proteins that have the same pi but varied MW and proteins that have the same MW but varied pi. Enzyme Assay Working with Proteins Once we purify out a protein or even during the purification itself, how do we know we are focused in on our protein of interest? ow do we know we have not thrown it out with our last step? For enzymes, we can use the reaction that it catalyzes to monitor the presence and amount of the enzyme throughout the purification To monitor this reaction, we need to design an assay that allows us to observe the catalytic effect of our enzyme. To develop an enzyme assay we must know the following: The overall equation for the catalyzed reaction An analytical procedure for determining the disappearance of substrate or appearance of product for the reaction Whether the enzyme requires co-factors such as metals or coenzymes for activity The dependence of the enzyme activity on substrate concentration The optimum p for activity A temperature zone in which the enzyme is stable and has high activity Chapter 3 47 Chapter

13 Working with Proteins Enzyme Activity One unit (1 U) of enzyme activity is defined as the amount of enzyme causing the transformation of 1.0 µmol of substrate per minute at 25 C. Activity is the total units of enzyme in a solution Specific Activity is the number of enzyme units per milligram of total protein. Activity Specific Activity Chapter 3 49 Protein Sequence The AA sequence of a protein represents very important information in determining its structure and function A human produces to proteins, each of which has a unique structure and sequence The AA sequence is important to the 3-D structure of the protein, which in turn, is important to the function of the protein. By knowing the AA sequence, we can make predictions about the 3-D structure, and the function of the protein. In addition, by comparing AA sequences for the same protein across species and within species, we can identify evolutionary changes and genetic defects which may cause disease Chapter 3 50 Protein Sequencing AA Composition Analysis In some cases, you may want to know the number of each type of amino acid within a polypeptide. The AA composition can be determined by completely hydrolyzing a protein under acidic conditions then analyzing the AAs using chromatography (usually via a detectable tag) Different amino acids in a peptide hydrolysate can be separated by ion-exchange chromatography on a sulfonated polystyrene Figure 4.18 from Biochemistry, 5 th Ed., Berg et al. resin (such as Dowex-50) Buffers (such as sodium citrate) of increasing p are used to elute the amino acids from the column. The amount of each amino acid present is determined from the absorbance. Aspartate, which has an acidic side chain, is first to emerge, whereas arginine, which has a basic side chain, is the last. In this case, the original peptide is revealed to be composed of one aspartate, one alanine, one phenylalanine, one arginine, and two glycine residues Chapter 3 51 Protein Sequencing Various procedures are available for determination of a protein s amino acid sequence. The Sanger Method The Edman Degradation Method Regardless of the technique used, the basic approach for sequencing proteins is similar: Separate the mature protein into individual polypeptide strands (if needed) Use at least two different chemical and/or enzymatic methods to break the polypeptide into two sets of smaller peptide segments Determine the sequence of each segment Reconstruct the overall sequence by ordering overlapping segments from the two sets Repeat the fragmentation step without breaking intra-strand connections to identify any Cys residues involved in disulfide bridges Chapter

14 Protein Sequencing Preliminary Steps The complete AA sequence of a protein includes the sequence of each of its subunits (if any), so these subunits must be identified and isolated Disulfide bonds within the protein structure must also be broken top separate and fully lineraize polypeptide chains Disulfide bonds can be reductively cleaved by treating them with 2-betamercaptoethanol (BME) or another mercaptan (contains a S group) N N C C 2 S S C 2 C O C O C BME + 2 SC 2C 2O The resulting free sulfhydral groups are then alkylated, usually by treatment with iodoacetate, to prevent reformation of the disulfide bond through O 2 oxidation 2 2 CYS C S + IC 2COO - CYS C S C 2COO - + I Chapter 3 53 N N C C 2 S + S C 2 C O C O C + SC 2C 2O SC 2C 2O Protein Sequencing Preliminary Steps Once the disulfide bonds are disrupted, the subunits are allowed to separate (if present) and the polypeptide(s) are linearized Now, N-terminal analysis can reveal the number of different subunits. Frederick Sanger developed the reagent 1-fluoro-2,4-dinitrobenzene (FDNB) for this purpose Other reagents include Dansyl chloride and Dabsyl chloride All of these reagents react with primary amines (the N-terminal amino group) For this procedure, the reagent is added to the protein solution resulting in a labeling of all amino terminii. The labeled polypeptide(s) are then hydrolyzed into individual AAs and the label is identified chromatographically. 3C C 3 N Dansyl Chloride Chapter 3 3C 54 3C N SO 2Cl N N SO 2Cl Dabsyl Chloride Protein Sequencing Edman Degradation If the polypeptide is small ( 50 AAs or less) you can determine the sequence using repeated cycles of Edman Degradation ere, Phenylisothiocyanate (PITC) reacts with the N-terminal amino group under alkaline conditions to form a phenylthiocarbamoyl (PTC) adduct. The peptide bond adjacent to this labeled terminus is cleaved under acidic conditions (Thifluoroacetic acid) This treatment cleaves only the peptide bond of interest! This derivative is then extracted in organic solvent and identified Protein Sequencing Polypeptide Cleavage If the polypeptide is larger than ~ 50 AAs, it must be cleaved, either chemically or enzymatically, into specific fragments that are small enough to be sequenced using Edman Degradation. Endopeptidases are enzymes that catalyze the hydrolysis of internal peptide bonds These enzymes are in the enzyme family of proteases and have side chain requirements for the residues flanking the target peptide bond (aka the scissle bond) These fragments are then separated using chromatography or electrophoresis Figure 4.24 from Biochemistry, 5 th Ed., Berg et al. Chapter 3 55 Figure 4.21 from Biochemistry, 5 th Ed., Berg et al. Chapter

15 Protein Sequencing Polypeptide Cleavage 3 N NMTQRCKPVNTFVEPLVDVQNVCFK COO - You must cleave the above peptide into smaller fragments. Which of the proteases listed would yield the most fragments? ow many? Which would yield the least fragments? ow many? Protein Sequencing Ordering Peptide Fragments Now that each fragment has been sequenced, the order of these fragments must be determined so that the entire polypeptide sequence can be established The AA sequence of the fragments formed from two separate cleavage methods are examined to identify overlapping sequences. Overlapping peptides can be identified and put in order Earlier identification of the Amino terminus can be used to identify the amino terminus fragment Chapter 3 57 Chapter 3 58 Protein Sequencing Ordering Peptide Fragments You wish to determine the sequence of a short polypeptide. Cleavage with trypsin yields three smaller peptide: L E Y N R Q A F V K Cleavage with chymotrypsin yields three peptides: Q A F N R L E V K Y What is the sequence of this polypeptide? Chapter 3 59 Protein Sequencing Locating Disulfide Bonds If the primary sequence includes disulfide bonds, their location is also of interest To determine the location of the disulfide bonds: Take a new sample of the protein and separate the polypeptides but DO NOT reduce the disulfide bridges Subject this strand to the same chemical or enzymatic cleavage technique as before and separate the fragments using electrophoresis Compare this pattern to that of the original sample If there is one disulfide bridge in your polypeptide, what would you expect the difference to be between the two samples? ow would you identify the location of the disulfide bridge? Chapter

16 Protein Sequencing Locating Disulfide Bonds Treatment of a polypeptide with 2-mercaptoethanol yields two polypeptides: A V C R T C K N F L Y K C F R T K C S Treatment of the intact polypeptide with trypsin yields fragments with the following AA composition A, R, C 2, S, V R, C 2,, K, T, F N, L, F, K, T K, T What are the positions of the disulfide bond(s)? Chapter 3 61 Protein Sequencing Chapter 3 62 Protein Sequencing and Mass Spectrometry Mass spectrometry has only recently become a tool of the biochemist This technique accurately measures the mass-to-charge ratio for ions in the gas phase New ionization techniques have allowed for the formation of macromolecule ions without degradation of the compound Matrix-assisted laser desorption/ionization (MALDI) Electrospray ionization (ESI) MS can be used to determined the molecular mass of a protein and to detect changes in this mass due to the presence of cofactors, metal ions, covalent modification due to reactions, etc. Tandem MS (MS/MS) can also be used to sequence short stretches of polypeptide. Chapter 3 63 Protein Synthesis Peptides are potentially useful as pharmacological agents making their production commercially profitable There are three ways to produce a peptide: Purification from the biological source enetic engineering Direct chemical synthesis R. Bruce Merrifield developed an innovative technique for peptide synthesis by attaching a growing polypeptide to an insoluble support and blocking the growing end Chapter

17 As stated before, the function of a protein depends on its 3-D structure, which, in turn, depends on its AA sequence The field of Molecular Evolution uses nucleotide and peptide sequences to explore evolution Basically, if two organisms are closely related, the sequences of their genes and proteins should be similar As organisms diverge evolutionarily, their sequences will also diverge We now have the computer power to store, translate and compare thousands of sequences, both peptide and nucleotide! Chapter Databases Containing ene and Protein Info After a protein s AA sequence has been determined, the information is usually deposited into a public database Several of these databases are available via the Internet and can be used to determine valuable information regarding proteins, nucleic acids and evolution Data Banks Containing Protein Sequences Swiss-Prot: Protein Info Resource: Protein Research Foundation: UniProt: Data Banks Containing ene Sequences enbank: European Bioinfor Institute: Chapter 3 66 There are some issues with this type of comparison, in that it is not as straight forward as just lining things up and calling it a match First, the AA residues required for function will usually be conserved across species while those residues of less importance may vary over time Second, there can be a rare transfer of a gene or group of genes from one species to another (lateral gene transfer). This may result in a false positive when running the sequence comparison. Can anyone think of an example of this lateral gene transfer? The study of molecular evolution generally focuses on families of closely related proteins, usually groups with functions that are essential to the viability of the organisms When comparing peptide sequences, you can find homologs, which can be either paralogs (in the same species) or orthologs (in different species). Chapter 3 67 A Blosum62 table (blocks substitution matrix complied from alignment that showed 62% identity) shows the scores assigned to AA substitutions within an alignment These substitutions can be conservative or nonconservative Remember that gaps in the sequence alignments incur penalties as well Chapter

18 In site directed mutagenesis experiments, ly is often successfully substituted for Val, but Val can rarely substitute for ly. Why? Below is a list of the first 10 residues of the B helix in Myoglobin from different species: Position uman Chicken Alligator Turtle Tuna Carp 1 D D K D D D 2 I I L L Y F 3 P A P S T E 4 E A T 5 M T Which positions appear unable to tolerate substitutions? Which can tolerate conservative substitutions? Which are highly variable? Chapter Q Q 8 E E E E L E 9 V V V V V V 10 L L I I L L Tree based on sequence divergence in the protein roel in bacteria The free end points of lines are external nodes and represent an extant species The points where two lines come together are internal nodes and represent extinct ancestor species The lengths of the lines connecting the nodes are proportional to the number of amino acid substitutions separating one species from another Chapter