Copy of Lecture Slides on General Biology IV - Biochemistry and Genetics PDF

There is a good chance we will not get through BI 214 General Biology IV all these slides on day one Biochemistry and Genetics weck A big thank you to Dr. Amy Connolly who developed the...

There is a good chance we will not get through BI 214 General Biology IV all these slides on day one Biochemistry and Genetics weck A big thank you to Dr. Amy Connolly who developed the course material for this class: Videos, quizzes, problems, etc And Dr. Alan Kelly who organized and selected much of the content we will cover Dr. Amy Connolly Dr. Alan Kelly  Today’s Objectives You should be able to… describe the basic molecular structure of an amino acid label the carbons in an amino acid’s side chain explain how proline and glycine’s structure differs determine whether an amino acid side chain is polar, non-polar or electrically charged explain how chemical polarity of a molecule affects its solubility in water, and apply this concept to amino acids  We will focus on the 20 “standard” amino acids that appear in our proteins and are encoded by our genes Unique amino acid structures and physical properties Unique Sequence (50- 2000 amino acids long) Unique Folding (three-dimensional shape) of the protein Tens of Thousands of Different Types of Proteins in Humans All Amino Acids Have an ⍺- Amino Group and an ⍺-Carboxylic Group; the R-group is the side chain and what makes them different ⍺ Proline’s Structure is an Exception when it comes to the amino group Proline ⍺ ⍺ Its R group is bound to its alpha-amino group, making it cyclic Its alpha amino group has one less H than the other amino acids. We will discuss later how this influences the three- dimensional shape of proteins! Glycine’s Structure is an Exception when it comes to the R group Glycine ⍺ ⍺ Its R group is a Hydrogen We will also discuss later how this influences the three-dimensional shape of proteins!  In reality, Amino Acids possess at least two charges in water (structure to the right) Which version is found in biological conditions? + - ⍺ ⍺ In water In a Vacuum (biological conditions) This form is not found in nature, but it might be found in quiz and exam questions zwitterion  Version 1 Version 2 Module Quiz 1-1 α β Question 1: γ The image below (to the right) shows an δ amino acid at physiological pH. Answer the ε following questions about this amino acid. a. How many carbons are in this amino acid’s side chain? b. Is the side chain of this amino acid ionized at physiological pH? c. At physiological pH, amino acids have at least two ionic charges, one positively charged group and one negatively charged group. What is the term for this sort of molecule?  Today’s Objectives You should be able to… describe the basic molecular structure of an amino acid label the carbons in an amino acid’s side chain explain how proline and glycine’s structure differs determine whether an amino acid side chain is polar, non- polar or electrically charged explain how chemical polarity of a molecule affects its solubility in water, and apply this concept to amino acids  Common to Categorize Amino Acids by the Polarity of their R-group Knowing the Polarity of the R-group Helps us predict where in the protein an amino acid might be located and who it’s interacting with! Which one is probably a transmembrane protein? a b Fiedler, Sebastian & Broecker, Jana & Keller, Sandro. (2010). Protein folding in membranes. Cellular and molecular life sciences : CMLS. 67. 1779-98. Module Quiz 1-1 Question 2: R- group a. Which amino acid has a polar side chain? b. Which amino acid is more soluble in water? c. Select the atom that serves as the hydrogen bond donor in the amino acid's R group Polarity and Water solubility ALL amino acids are soluble in water, we are just comparing the R groups The more polar a compound the more soluble it is in water. What makes a covalent bond polar versus non-polar?  Polarity Refers to the Distribution of Electrons around the molecule Cl2 HCl Cl Cl δ+ H Cl δ- Nonpolar covalent bond Polar covalent bond Bonding electrons shared equally Bonding electrons shared between two atoms. unequally between two atoms. No partial charge on atoms Partial charge on atoms  Polar bonds often result from an electronegative differences between two atoms in a bond O-H bond Electronegativity: an atom’s attraction for - = 1.4 the electrons in a covalent bond. The more electronegative an atom, the more strongly it Polar covalent pulls shared electrons toward itself C-H bond - = 0.4 https://www.brainkart.com/article/Electronegativity_34694/ Non-polar covalent Hydrocarbons like methane Non-polar covalent bonds: Polar covalent bonds: Ionic bonds: EN difference < 0.5 EN difference 0.5-2.1 EN difference > 2.1 Polar molecules can better disperse in water because they can interact with water molecules. Hydrogen bonds are a kind of dipole-dipole interaction Hydrogen bond Hydrogen bond: H atom covalently bonded to N, O, or F (Hydrogen bond donor) attracted to the free electron pair of another N, O, or F (Hydrogen bond acceptor)  Intermolecular Forces- Relative Strengths Intermolecular Forces Strongest to weakest: 1. Ionic Attraction ( + and -) O 2. Ion-Dipole ( + and 𝛿𝛿-) or (- and 𝛿𝛿+) K+Cl- H H --- 3. Dipole-Dipole (𝛿𝛿 + and 𝛿𝛿-) O H H Hydrogen Bond is the strongest of these 4. Van der waals forces Ion-Dipole between non-polar molecules; results from temporary dipoles  Nonpolar Side Chains Remember that ALL amino acids are soluble in water we are just looking at the relative solubility of the side chains! Glycine is often grouped in with the nonpolar side chains, but since its R group is just a Hydrogen it doesn’t make sense to describe its polarity  Polar Side Chains R groups contain electronegative atoms creating an asymmetrical pull of electrons least polar Serine, Threonine and Tyrosine all contain hydroxyl groups Asparagine and Glutamine contain amides Cysteine contains a sulfhydryl group (weakly polar, and sometimes categorized with non-polar category)  Electrically Charged Side Chains Side Chains are Charged at Neutral pH Is this an alpha carboxyl group? Aspartic Acid Glutamic Acid Conjugate Base Shown Conjugate Acid Shown Asparartic Acid/Aspartate and Glutamic Acid/Glutamate have carboxyl groups Lysine has an amino group and Arginine has a guanidino group Histidine has an imidazole ring; (Only 10% are charged at pH7)  Would the answer change in part Module Quiz 1-1 (b) if we were comparing it to Aspartic Acid’s R group? Question 2: a. Which amino acid has a polar side chain? b. Which amino acid is more soluble in water? c. Select the atom that serves as the hydrogen bond donor in the amino acid's R group Aspartate (D, Asp) Generally speaking (if other components of the structure are pretty similar) charged groups will be more soluble than polar groups  Hydrophobicity Tables are Based on Experimental Data that Give us an Idea of How Soluble a Side Chain is Hydrophobicity Table of Side Chains ΔG H2O → octanol–water partition coefficient 2. Determine ∆G for movement octanol of a.a. from water to Octanol transfer Trp -2.09 Least Soluble in Water = -ΔG non-polar Phe -1.71 Leu -1.25 Ile -1.12 Octanol Tyr -0.71 Met -0.67 (non-polar) Val -0.46 Remember that ALL amino Cys -0.02 Pro 0.14 acids are soluble in water; 1. Add octanol polar Thr 0.25 we are just looking at the Ser 0.46 relative solubility of the side Ala 0.5 chains! Gln 0.77 Water Asn 0.85 (polar) polar-charged Gly 1.15 Arg+ 1.81 His+ 2.33 - ∆G  exergonic  more hydrophobic Lys+ 2.8 Glu- 3.63 + ∆G  endergonic  less hydrophobic Asp- 3.64 Most Soluble in Water = +ΔG  Though not perfectly, you can see the trends we discussed reflected in real measurements Hydrophobicity Table of Side Chains ΔG H2O → octanol transfer Trp -2.09 Least Soluble in Water non-polar Phe -1.71 Size also Affects an Amino Leu -1.25 Acid’s Solubility in Water Ile -1.12 Tyr -0.71 Met -0.67 Val -0.46 Less Soluble Cys -0.02 in Water Pro 0.14 polar Thr 0.25 Ser 0.46 Ala 0.5 Gln 0.77 Asn 0.85 polar-charged Gly 1.15 Arg+ 1.81 His+ 2.33 Lys+ 2.8 Glu- 3.63 More Soluble Asp- 3.64 Most Soluble in Water in Water  Though not perfectly, you can see the trends we discussed reflected in real measurements Hydrophobicity Table of Side Chains ΔG H2O → General trend octanol transfer Trp -2.09 Least Soluble in Water Least H2O soluble non-polar Phe -1.71 Leu -1.25 Ile -1.12 Large non-polar Tyr -0.71 Met -0.67 Small non-polar Val -0.46 Cys -0.02 Polar Pro 0.14 polar Thr 0.25 Ser 0.46 Charged Ala 0.5 Gln 0.77 Most H2O soluble Asn 0.85 polar-charged Gly 1.15 Arg+ 1.81 His+ 2.33 Lys+ 2.8 Glu- 3.63 Asp- 3.64 Most Soluble in Water  Problem Set: Chapter 2; Question 9 9. Rank the following R groups in increasing hydrophobicity (1: lowest; 5: highest) Disclaimer: There’s not a “perfect answer,” but there are correct trends! You just need to understand general trends!   Practice Question! On the molecules below draw the H-bond acceptors and Donors. Review Acid/Base Chemistry and Apply to Amino Acid Chemistry  Course Business Reminder: No lab this week Next week you will have amino acid flash cards to bring to lab (please see instructions in Pre-lab Quiz) Problems with Canvas? Help desk in PLC Room 68 open 9-4 Monday through Friday. Telephone support is also available at 541-346-4357 (6-HELP) Hydrophobicity Tables are Based on Experimental Data that Give us an Idea of How Soluble a Side Chain is Hydrophobicity Table of Side Chains ΔG H2O → octanol–water partition coefficient octanol transfer Trp -2.09 Phe -1.71 Leu -1.25 Ile -1.12 Tyr -0.71 Met -0.67 Val -0.46 Remember that ALL amino Cys -0.02 Pro 0.14 acids are soluble in water; Thr 0.25 we are just looking at the Ser 0.46 relative solubility of the side Ala 0.5 chains! Gln 0.77 Asn 0.85 Gly 1.15 Arg+ 1.81 His+ 2.33 Lys+ 2.8 Glu- 3.63 Asp- 3.64  Hydrophobicity Tables are Based on Experimental Data that Give us an Idea of How Soluble a Side Chain is Hydrophobicity Table of Side Chains ΔG H2O → octanol–water partition coefficient octanol transfer Trp -2.09 Least Soluble in Water = -ΔG non-polar Phe -1.71 Leu -1.25 Ile -1.12 Tyr -0.71 Met -0.67 Val -0.46 Cys -0.02 Pro 0.14 polar Thr 0.25 Ser 0.46 Ala 0.5 Gln 0.77 Asn 0.85 polar-charged Gly 1.15 Arg+ 1.81 His+ 2.33 - ∆G  exergonic  more hydrophobic Lys+ 2.8 Glu- 3.63 + ∆G  endergonic  less hydrophobic Asp- 3.64 Most Soluble in Water = +ΔG  Though not perfectly, you can see the trends we discussed reflected in real measurements Hydrophobicity Table of Side Chains ΔG H2O → octanol transfer Trp -2.09 Least Soluble in Water non-polar Phe -1.71 Size also Affects an Amino Leu -1.25 Acid’s Solubility in Water Ile -1.12 Tyr -0.71 Met -0.67 Val -0.46 Less Soluble Cys -0.02 in Water Pro 0.14 polar Thr 0.25 Ser 0.46 Ala 0.5 Gln 0.77 Asn 0.85 polar-charged Gly 1.15 Arg+ 1.81 His+ 2.33 Lys+ 2.8 Glu- 3.63 More Soluble Asp- 3.64 Most Soluble in Water in Water  Though not perfectly, you can see the trends we discussed reflected in real measurements Hydrophobicity Table of Side Chains ΔG H2O → General trend octanol transfer Trp -2.09 Least Soluble in Water Least H2O soluble non-polar Phe -1.71 Leu -1.25 Ile -1.12 Large non-polar Tyr -0.71 Met -0.67 Small non-polar Val -0.46 Cys -0.02 Polar Pro 0.14 polar Thr 0.25 Ser 0.46 Charged Ala 0.5 Gln 0.77 Most H2O soluble Asn 0.85 polar-charged Gly 1.15 Arg+ 1.81 His+ 2.33 Lys+ 2.8 Glu- 3.63 Asp- 3.64 Most Soluble in Water  Problem Set: Chapter 2; Question 9 9. Rank the following R groups in increasing hydrophobicity (1: lowest; 5: highest) Disclaimer: There’s not a “perfect answer,” but there are correct trends! & + 2 + th - 1 43 I least polar +3 ring + 3 +U b ↑ +7 + 3 + 1 34 5 2  Conclusion All amino acids alone are polar and soluble in water But their side chains range from non-polar and less soluble in water, to polar or polar-charged and more soluble in water Understanding their properties helps a biochemist to understand the interactions that occur within a protein  Today’s Objectives You should be able to… explain what the Bronsted-Lowry definition of an acid/base is, and what makes one strong versus weak describe what an acid-dissociation equation is, identify the acid and conjugate base in the reaction and understand how the equilibrium constant (Ka) and its pKa vary by acid strength identify the spot on the titration curve where the pKa lies and know its significance explain how [HA]:[A-] changes with pH use the Henderson-Hasselbalch equation to calculate, at a specific pH, the ratio or fraction of [HA] and [A-].  HA = conjugate acid Brønsted-Lowry acid dissociation HA A- + H+ A- = conjugate base useful recap acid dissociation [A-] [H+] constant Ka = [HA] Weak acid Strong acid pKa = -log Ka HA A- + H+ HA A- + H+ Ka = [A-] [H+] [A-][H+] Ka = [HA] [HA] Ka < 1 Ka > 1 pKa is positive pKa is negative Module Quiz 1-2 Question 1 A B A B pKa: -8 Version 1 pKa: 4.2 Version 2 B A B A Answer the following questions for each version: a. Which compound in the forward direction of the acid-dissociation equation acts as the Bronsted-Lowry acid? 1. A (H (Ou7 2 B (C6HsCOOlt). b. Is the Ka of the acid greater than or less than 1?. greater 1 negative pra (strong acid). less 2 than positive pha (weak acid) c. Are the products or reactants favored? 1. products 2 reactants.  Today’s Objectives You should be able to… explain what the Bronsted-Lowry definition of an acid/base is, and what makes one strong versus weak describe what an acid-dissociation equation is, identify the acid and conjugate base in the reaction and understand how the equilibrium constant (Ka) and its pKa vary by acid strength identify the spot on the titration curve where the pKa lies and know its significance explain how [HA]:[A-] changes with pH use the Henderson-Hasselbalch equation to calculate, at a specific pH, the ratio or fraction of [HA] and [A-]. Module Quiz 1-2 Question 2 Below is the titration curve for a monoprotic weak acid/weak base. In order to produce this curve, you titrate the weak acid with NaOH until its all been converted to its weak base form. Recall from general chemistry, at 1.0 equivalents of titrant (OH-), all of your original acid analyte has been neutralized (deprotonated) and is in its basic form. a. What is the pKa of this compound? 4 equivalent ↑ pH b. At a pH of 6 will the concentration of the conjugate base be higher or lower than the PKa where the meet concentration of conjugate acid? ↑ higher ·  At 0 base equivalents added the analyte (compound of interest): monoprotic buffer in this case is fully protonated  100% in its conjugate acid (HA) form Proton Anion H+ A- Initially as OH equivalents are HA = conjugate acid A- = conjugate base added PH increases rapidly outside of the buffering range of the analyte H A- H H A- -H A- H A- H A A- H H A- A- H A- A-H H H A- A- When the pH enters the buffering rage the analyte starts losing protons. H A- H H A- -H A- H A- H A A- H H A- A- H A- A-H H H A- A- In the buffering rage the pH doesn’t change as much with OH equivalents added because the analyte starts losing protons (instead of hydronium ions losing them) H A- H H A- H A- A- A- A- H A- -H H A -H A- H A A- A- When half of the analyte is in the conjugate acid form and half is in the conjugate base form (½ the equivalency point) the pH = pKa. pKa = pH when [HA] = [A-] pKa H A- H A- H A- A- A- A- H A- A- H A- A- H A- A- After the equivalency point the pH changes rapidly with additional OH equivalents added A- A- A- A- A- A- A- A- A- A- A- A-  Analyte more deprotonated pH = pKa More conjugate base form [HA] = [A-] carboxyl acid group O [HA] < [A-] O R C pH > pKa R C OH O H H R N H+ pH < pKa R N H H [HA] > [A-] More conjugate acid form Analyte more protonated Take away points Module Quiz 1-2 Question 2 Below is the titration curve for a monoprotic weak acid/weak base. In order to produce this curve, you titrate the weak acid with NaOH until its all been converted to its weak base form. Recall from general chemistry, at 1.0 equivalents of titrant (OH-), all of your original acid analyte has been neutralized (deprotonated) and is in its basic form. c. Using the pKa you calculated in part a, ver1 : 80%. find the percent of molecules that are in Ver 2 6 : % the conjugate acid form at a pH of 3.4 (version 1) and pH of 5.2 (version 2). Entry 1 of HA for every ly Of A- · format: 2 sig figs [A - ] zPH cog[A] S z= 4 109 + · Ver z CHA] PH = pku. +. Y Y [HA] - 2) PKU & - [A ] - 1) ph [A-] 1 2 = 109 CHA] Ver 1 3 4= 4... + 109 [HA] more than So1. since more acid [A - y - Y 1012 = - 0 6. = 10 - 0 0. = [A -) CHA] -CA 0 25 (A J [HA] i -. = - I for every 0 25 base there are I acid [HA]. CAT 0 06 6 HA] = [HA] 23 +. 10-0 6 100 CATHA]. = 6 0 = 80%.. How do you know when to use the Henderson-Hasselbach equation? When it’s NEAR the pKa! within 2 pH units for this class Ratio Percent Base : Acid Base & Acid pH= pKa+3 1000 : 1 99.9% 0.1% Use HH Equation pH= pKa+2 100 : 1 99% 1% pH= pKa+1 10 : 1 91% 9% pH= pKa 1 : 1 50% 50% pH= pKa-1 1 : 10 9% 91% pH= pKa-2 1 : 100 1% 99% pH= pKa-3 1 : 1000 0.1% 99.9%  We will be applying Acid/Base Chemistry to Amino Acids, which are diprotic (Can donate Protons Twice) [HA] [A-] pKa ~2 + + - ⍺ ⍺ pKa ~9-10 + - ⍺ - ⍺ [A-] [HA] Question: Which group is a stronger acid? alpha-amino or alpha- carboxylic? pKa ~2 + + - ⍺ ⍺ pKa ~9-10 + - ⍺ - ⍺  & - +1 +0.5 0 -0.5 -1 M a.a. charge  Rotate 900 We will be working with the effects of pH on the charge of a.a. So we rotate the graph 900 to show charge as a function of pH. +1 +0.5 0 -0.5 -1 a.a. charge  pK2 pK1 = = +1 +0.5 0 -0.5 -1 a.a. charge  files Keys Problem Set: Chapter 1, Question 4 4. Consider the following two carboxylic acids: a) Which form (conjugate acid or conjugate base) is shown for each? b) Draw the complementary acid or base form for each. c) In water at pH 7, which form will predominate for each? d) Which carboxylic acid will be more dissociated in water at pH 7?  Problem Set: Chapter 1, Question 5 Consider the following two cyclic amines: a) Which form (conjugate acid or conjugate base) is shown for each? b) Draw the complementary acid or base form for each. c) In water at pH 7, which form will predominate for each? Amino Acids with Nitrogen in their side chain: Remember the pattern! Nitrogen-Acidic Form Nitrogen-Basic Form N N : H+ 4 bonds 3 bonds 0 lone pair 1 lone pair +1 charge no charge Amino Acids with Nitrogen in their side chain: Remember the pattern!  Problem Set: Chapter 1, Question 6 Each of the following molecules is dissolved in buffered solutions of: (a) pH = 2 and (b) pH = 11. For each indicate the solution in which the charged species will predominate. The underlined atom is dissociable (assume that the molecules do not appreciably change the pH of the solution). week 2 was not in class Diprotic Amino Acids +  Course Business Turn in your pre-lab quiz by 11:59 on Wednesday make your flashcards for all 20 amino acid for Lab 1 (instructions found in pre-lab quiz)  Objectives You should be able to… identify the acid/base forms for the α-amino and α-carboxylic groups of an amino acid determine which amino acid structure predominates at a given pH determine the charges of the individual ionizable side chains as well as the net charge at a given pH calculate the isoelectric point (pI) of a diprotic amino acid use the Henderson-Hasselbalch equation to calculate the average charge of an ionizable group when the pH falls near its pKa – recognize when it needs to be performed when calculating the average net charge of an amino acid A Question: Which group/s (A- + D) predominates at which point (1-5)? R B 5 4 R C 3 - 2 1 R D + - First, which form would we R not find on our graph? pKa Table for the Amino Acids Amino Acid Group* pK1 pK2 pK3 (a-carboxyl) (a-amino) (side chain) Alanine (Ala) Nonpolar 2.4 9.9 Arginine (Arg) Charged 1.8 9.0 12.5 Asparagine (Asn) Polar 2.1 8.7 Aspartic acid (Asp) Charged 2.0 9.9 3.9 Cysteine (Cys) Polar 1.9 10.7 8.4 Glutamic acid (Glu) Charged 2.1 9.5 4.1 Glutamine (Gln) Polar 2.2 9.1 Glycine (Gly) Nonpolar 2.4 9.8 Histidine (His) Polar 1.8 9.3 6.0 Isoleucine (Ile) Nonpolar 2.3 9.8 Leucine (Leu) Nonpolar 2.3 9.7 Lysine (Lys) Charged 2.2 9.1 10.5 Methionine (Met) Nonpolar 2.1 9.3 Phenylalanine (Phe) Nonpolar 2.2 9.3 Proline (Pro) Nonpolar 2.0 10.6 Serine (Ser) Polar 2.2 9.2 Threonine (Thr) Polar 2.1 9.1 Tryptophan (Trp) Nonpolar 2.5 9.4 Tyrosine (Tyr) Polar 2.2 9.2 10.5 Valine (Val) Nonpolar 2.3 9.7  Module Quiz 1-3: Question 1 Version 1 Version 2 At a low pH the overall charge on a At a high pH the overall charge on a diprotic amino acid is more diprotic amino acid is more positive/negative than the overall positive/negative than the overall charge at a higher pH. charge at a lower pH. As the pH increases from a pH of As the pH decreases from a pH of zero, the alpha carboxylic/alpha 14, the alpha carboxylic/alpha amino is the first group of a diprotic amino is the first group of a diprotic amino acid to lose a proton. This amino acid to gain a proton. This group goes from a charge of 0/+1/-1 group goes from a charge of 0/+1/-1 to a charge of 0/+1/-1 to a charge of 0/+1/-1  Consider How the Structure Changes: Net Charge of Amino Acid Changes with pH pH Below ~2 pH ~2 to ~9-10 pH Above ~9 -10 Net: +1 0 -1 +1 0 +1 -1 0 -1 pKa pKa ~2 ~9-10 + + - - R R R  We have been looking at “Equivalents vs. pH” 10 12 14 net charge 8 6 4 2 0 pH +1 +0.5 0 -0.5 -1.0 pKas found in the middle of horizontal flattenings (where pH changes slows)  Now Consider the titration curve “pH vs Net Charge” +1 +0.5 Question: Next to the net charge tick marks, fill in the net- 0 charges on the y-axis. -0.5 -1.0 0 2 4 6 8 10 12 14 pH pKas will now be found in the middle of vertical flattenings (again, where pH changes slows) Answer Now Consider the titration curve “pH vs Net Charge” +1 +0.5 net charge Notice that when the pKa= pH the pI: pH where charge net charge of a diprotic amino acid 0 is neutral is +0.5 for pKa1 and -0.5 for pKa2. -0.5 -1.0 0 2 4 6 8 10 12 14 pH pKas will now be found in the middle of vertical flattenings (again, where pH changes slows)  Objectives You should be able to… identify the acid/base forms for the α-amino and α-carboxylic groups of an amino acid determine which amino acid structure predominates at a given pH determine the charges of the individual ionizable side chains as well as the net charge at a given pH calculate the isoelectric point (pI) of a diprotic amino acid use the Henderson-Hasselbalch equation to calculate the average charge of an ionizable group when the pH falls near its pKa; – recognize when it needs to be performed when calculating the average net charge of an amino acid  Module Quiz 2-1: Question 2 Isoleucine pKa 9.76 pKa 2.32 The diprotic amino acid shown above is how it’d appear in a vacuum. a. What is isoelectric point? (Round your answer to two decimal places, for example: 0.13 or 1.45 or 3.30).  Module Quiz 2-1: Question 2: Answer Isoleucine pKa 9.76 pKa 2.32 The diprotic amino acid shown above is how it’d appear in a vacuum. a. What is isoelectric point? (Round your answer to two decimal places, for example: 0.13 or 1.45 or 3.30). pI 6.04 Module Quiz 2-1: Question 2 Version 1 The diprotic amino acid isoleucine is shown below as it would appear in a vacuum. pKa 9.76 pKa 2.32 b. What is the average net charge of the amino acid at a pH 2.45? Round to two decimals; include charge. Format example: (+0.13 or -1.45 or -3.30). Do we have to use the Henderson-Hasselbach equitation to calculate the charge on the: Amino group? Carboxyl group? What is the average net charge of the pKa 9.76 pKa 2.32 amino acid at a pH 2.45? Module Quiz 2-1: Question 2 Version 1 The diprotic amino acid isoleucine is shown below as it would appear in a vacuum. pKa 9.76 pKa 2.32 b. What is the average net charge of the amino acid at a pH 10? Round to two decimals; include charge. Format example: (+0.13 or -1.45 or -3.30). Do we have to use the Henderson-Hasselbach equitation to calculate the charge on the: Amino group? Carboxyl group? What is the average net charge of the pKa 9.76 pKa 2.32 amino acid at a pH 10? how do you know if you need to use the numerator or the denominator at the end of the HH equation. Conjugate base= [A-] [NH2] [COO-] Conjugate acid= [HA] [NH3+] [COOH] You always want to find out what % of the group is in the charged form For the alpha-amino group, the charged form is the conjugate acid (NH3+), so we need to solve for the denominator For the alpha-carboxy group, the charged form is the conjugate base (COO-), so we need to solve for the numerator Remember, these questions ask for the net charge at a particular pH so you need to know if each group provides a – (alpha-C) or a + (alpha-N) charge, and what % of each group is in the charged form (HH equation).  Triprotic Amino Acids (Our Next Topic!) In addition to having an alpha amino and alpha carboxylic group, triprotic amino acids also can gain/lose a proton on their R group. pKa2 pKa1 + R pKa3 This pKa3 (the R group’s pKa) varies widely among the amino acids R group pKa2 pKa3 pKa2 R group pKa3 pKa1 pKa1 +1 0 -1 -2 charge This pKa3 (the R group’s pKa) varies widely among the amino acids R group pKa2 pKa3 pKa2 R group pKa3 pKa1 pKa1 +1 0 -1 -2 +2 +1 0 -1 charge charge Triprotic Amino Acids Question: Without looking at the next slide, consider what you’ve learned about acid/base chemistry so far, and fill in the blanks on this table by drawing the structure for the missing conjugate acid or base. Triprotic Amino Acids Attention! Be able to identify all of these structures Know how they are protonated and deprotonated Memorize these pKas only (pKa of R groups only) Note: Should also know proline and glycine’s structure  Key Functional Groups to Know and be able to Apply Acid Form [HA] Base form [A-] Example Structure Charge Structure Charge −NH3+ +1 −NH2 0 ɑ-amino R-group of Lys =NH2+ +1 =NH 0 R-group of Arginine ≡NH+ +1 ≡N 0 R group of His COOH 0 COO- -1 ɑ- COOH R-groups of Asp and Glu OH 0 O- -1 R-group of Tyrosine SH 0 S- -1 R-group of Cysteine  Histidine’s Structure 1. Either Nitrogen can accept the proton due to resonance (double bonds will change in drawing) 2. You will NOT see the following structure (2 Protons on one Nitrogen) H--- + Do not draw this! Triprotic Amino Acids- Next Question pH 6 Question: At a pH of 6, estimate the charge of the side chain only for each amino acid. Circle the form/s that predominate at this pH.  Question: To the left is a structure of Cysteine in a vacuum. Determine the average net pKa1 1.9 pKa2 10.7 charge at a pH of 8.0. Fill out the table to guide you. Charge at pH of 8.0 pKa (R) 8.4 ⍺-carboxylic R- sulfhydrl ⍺- amino group NET Determine the average net pKa1 1.9 pKa2 10.7 charge at a pH of 8.0. pKa (R) 8.4 Charge at pH of 8.0 ⍺-carboxylic R- sulfhydrl ⍺- amino group NET  Problem Set: Chapter 1, Question 6 Each of the following molecules is dissolved in buffered solutions of: (a) pH = 2 and (b) pH = 11. For each indicate the solution in which the charged species will predominate. The underlined atom is dissociable (assume that the molecules do not appreciably change the pH of the solution). iClicker responses and participation will not be graded iClicker: Log in using iClicker student app or going to iclicker.com Click: “sigh in”  student To add a class, go to + sign in upper right-hand corner Institution: University of Oregon Search for “BI 214” Click join button Click on course name to participate Triprotic Amino Acids and Polypeptides Course Business Pre-lab due by end of day Post-lecture quiz from last wed also due by end of day Print out the lab for this week and bring it with you to lab Bring amino acid cards to lab this week! Please make sure you stay on top of due dates Today’s Objectives Part 1: Triprotic Amino Acids You should be able to: identify an amino acid by its R group, know the pKa of the R group, and know the acid/base forms of the R groups recognize (or narrow down) the triprotic amino acid you are looking at if given the plot of its curve pH vs charge determine which amino acid structure predominates at a given pH calculate the isoelectric point (pI) explain how the charge changes with pH apply your ability to use the Henderson-Hasselbalch equation to triprotic amino acids, in order to calculate the charges of their three ionizable groups and the net charge. Triprotic Amino Acids Key Functional Groups to Know Charge of R group at pH 6 Acid Form [HA] Base form [A-] Structure Charge Structure Charge COOH 0 COO- -1 OH 0 O- -1 SH 0 S- -1 −NH3+ +1 −NH2 0 =NH2+ +1 =NH 0 ≡NH+ +1 ≡N 0 Nitrogen-Acidic Form Nitrogen-Basic Form 4 bonds 3 bonds 0 lone pair H+ 1 lone pair +1 charge N no charge N A. Acid O Base B.  Histidine’s Structure 1. Either Nitrogen can accept the proton due to resonance (double bonds will change in drawing) 2. You will NOT see the following structure (2 Protons on one Nitrogen) H--- + Do not draw this! Today’s Objectives Part 1: Triprotic Amino Acids You should be able to: identify an amino acid by its R group, know the pKa of the R group, and know the acid/base forms of the R groups recognize (or narrow down) the triprotic amino acid you are looking at if given the plot of its curve pH vs charge calculate the isoelectric point (pI) explain how the charge changes with pH determine which amino acid structure predominates at a given pH apply your ability to use the Henderson-Hasselbalch equation to triprotic amino acids, in order to make calculations charges about any of their three ionizable groups and the net charge. This pKa3 (the R group’s pKa) varies widely among the amino acids depends on amino acid could be > PKA2 PKa , -R group - PKa , -praz -> R group (pKaz) Al Al alpha amino R group pKa2 alpha amino pKa3 pKa2 R group pKa3 Alpha carboxy ( pKa1 pKa1 +1 0 -1 -2 charge This pKa3 (the R group’s pKa) varies widely among the amino acids R group pKa2 pKa3 pKa2 R group pKa3 pKa1 pKa1 +1 0 -1 -2 +2 +1 0 -1 charge charge  Identifying triprotic a.a. from the pH vs Charge curve Identify the pKa’s Use the charge range to narrow down the R group possibilities Remember that pKa3 can be inbetween pKa1 and pKa2, or after pKa2 A. COO-/COOH B. NH2/NH3+ 6 Module Quiz 2-2, Question 1 The pH vs charge graph for a triprotic amino acid is shown below. Please answer the following questions about the amino acid. a. Which of the following triprotic amino acids is this? Version 1 Version 2 · pKa ! o pKa , · pKa3 ? 8 S PKazOrpKaz = ·. · PKAz · or PKaz PKaz = 10. S Answer Options Answer Options Lysine Lysine Arginine Arginine Glutamic Acid Histidine 6 0. Histidine Aspartic Acid Cysteine Tyrosine  Cysteine’s Curve Can Be Hard to Distinguish from Tyrosine Cysteine Tyrosine 1 +1 * * pKa1 0.5 pKa1 +0.5 0 0 Net Charge net charge -0.5 pKa3(R)(8.4) -0.5 pKa2 (9.2) 0 -1 pKa3(R)(10.5) -1.5 pKa2 (10.7) -1.5 -2 -2 0 2 4 6 8 10 12 14 pH Cysteine pH 0 Tyrosine pH 0 +1 +1 ---H+  But Tyrosine and Lysine Can be Distinguished By looking at the Charges on the Y-axis! Lysine Tyrosine A B 1 1 +2 0.5 pKa1(2.2) 0.5 pKa1 (2.2) +1.5 +1.00 0 Net Charge net charge Net Charge -0.5 +0.5 pKa2 (9.1) -0.5 pKa2 (9.2) 0-1 -1 -0.5 -1.5 pKa1 (10.5) pKaR (10.5) -1.5 -1.0-2 -2 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 pH pH Lysine Tyrosine Which one is Tyrosine?  Triprotic Amino Acid Titration Curve Overview Histidine Lysine Arginine positively charged pKa1 pKa1 pKa1 pKa3 (6.0) pKa2 R groups pKa2 pKa3 (10.5) pKa3 (12.5) pKa2 Asp/Glu Cysteine Tyrosine negatively charged R groups pKa1 pKa1 pKa1 pKa3 (~4) pKa3 (8.4) pKa2 pKa2 pKa3 (10.5) pKa2 Today’s Objectives Part 1: Triprotic Amino Acids You should be able to: identify an amino acid by its R group, know the pKa of the R group, and know the acid/base forms of the R groups recognize (or narrow down) the triprotic amino acid you are looking at if given the plot of its curve pH vs charge calculate the isoelectric point (pI) explain how the charge changes with pH determine which amino acid structure predominates at a given pH apply your ability to use the Henderson-Hasselbalch equation to triprotic amino acids, in order to calculate the charges of their three ionizable groups and the net charge.  First: Find an Estimate Using the Graph Module Quiz 2-2, Question 1b Second: Calculate to Find the Precise Value The pH vs charge graph for a triprotic amino acid is shown below. Please answer the following questions about the amino acid. b. What is the isoelectric point? Version 1: + 10 - I Version 2: ↓ 1.9 ↓ ↓ - 8.4 10 7 pro. o deprogranted - NHz entrtdrpro look ted O charge + 1 & PI [ S 2. 6 7 - PI find O , that ~ 7 % isoelectric point  First: Find an Estimate Using the Graph Module Quiz 2-2, Question 1b Second: Calculate to Find the Precise Value The pH vs charge graph for a triprotic amino acid is shown below. Please answer the following questions about the amino acid. b. What is the isoelectric point? Version 1:  First: Find an Estimate Using the Graph Module Quiz 2-2, Question 1b Second: Calculate to Find the Precise Value The pH vs charge graph for a triprotic amino acid is shown below. Please answer the following questions about the amino acid. b. What is the isoelectric point? - I Version 2: 10 S 6 0. 99. 3 J 200 - EN NHz NH zt Cool ENH + 1 7. Today’s Objectives Part 1: Triprotic Amino Acids You should be able to: identify an amino acid by its R group, know the pKa of the R group, and know the acid/base forms of the R groups recognize (or narrow down) the triprotic amino acid you are looking at if given the plot of its curve pH vs charge calculate the isoelectric point (pI) explain how the charge changes with pH determine which amino acid structure predominates at a given pH apply your ability to use the Henderson-Hasselbalch equation to triprotic amino acids, in order to calculate the charges of their three ionizable groups and the net charge. For example, your Module Quiz 2-2, Question 1c The pH vs charge graph for a triprotic amino acid is shown below. Please answer the following questions about the amino acid. c. What form of this amino acid dominates at a pH of 14 for Version 1 and a pH of 4.5 for Version 2? Version 1: pH of 14 Version 2: pH of 4.5 deproganated pKa2 9.3 pKa1 1.9 pKa2 10.7 pKa1 1.8 pKa (R) 6.0 pKa (R) 8.4 Today’s Objectives Part 1: Triprotic Amino Acids You should be able to: identify an amino acid by its R group, know the pKa of the R group, and know the acid/base forms of the R groups recognize (or narrow down) the triprotic amino acid you are looking at if given the plot of its curve pH vs charge calculate the isoelectric point (pI) explain how the charge changes with pH determine which amino acid structure predominates at a given pH apply your ability to use the Henderson-Hasselbalch equation to triprotic amino acids, in order to calculate the charges of their three ionizable groups and the net charge. Example Question: Work on your Own After Class; like other problems we’ve done To the left is a structure of Cysteine in a vacuum. Determine the average net charge at a pH of 8.0. Fill pKa1 1.9 pKa2 10.7 out the table to guide you. Charge at pH of 8.0 ⍺-carboxylic pKa (R) 8.4 R- sulfhydrl ⍺- amino group NET Polypeptides Today’s Objectives Part 2: Polypeptides You should be able to… label the peptide bond, the alpha carbon, and the carbonyl and amide groups in a polypeptide; also be able to label these in a skeletal structure of a polypeptide calculate the pI of a polypeptide determine how the pI of a polypeptide would be affected if one residue were to be changed. Module 1-4 Question 2 a. Match the letter labels to the appropriate components of a polypeptide. i) carbonyl group B ii) amide C iii) alpha amino group A iv) alpha carboxylic group E v) peptide bond D b. How many peptide bonds are in this polypeptide? ___ 3 Residues? ____ 4  Module 1-4 Question 2 c. If the second residue were replaced with arginine would the pI increase or decrease? First: Answer this question by explaining it conceptually. pKa 9.7 Second: Answer the question by calculating the pKa 2.1 pI of the new polypeptide and comparing it to the original. pKa 6.0 Valine-Isoleucine-Histidine- Threonine pI 7.85 Valine-Arginine-Histidine- Threonine ? First Approach: Consider it Conceptually The Rule of Thumb pI increases when the side chain’s charge increases. Any of the following: 0 + -+ -0 pI decreases when the side chain’s charge decreases. Any of the following: 0 - +- +0 *Non-ionizable side chains are neutral (0)  pI calculation pKa 12.5 2.1 6.0 9.7 12.5 ⍺-COO- His(R) NH+ ⍺-NH3+ pKa 9.7 Arg(R) NH2+ pKa 2.1 Net pKa 6.0 Valine-Isoleucine-Histidine- Threonine pI 7.85 Valine-Arginine-Histidine- Threonine ? Isoelectric Point (pI) and solubility Effect of pH on milk protein (casein) Maureen Daschel: https://www.youtube.com/watch?v=egazCtStwsQ The solubility of the amino acid or protein is the lowest at its pI  Isoelectric Point (pI) of a Polypeptide- Practical Use Isoelectric Focusing: Separates proteins by their isoelectric point! solution of molecules applied to the gel. More positive molecules below their pI, attracts cations attracts anions more negative above the pI Anions drawn to anode; cations drawn to cathode eventually stall when they hit their pI (neutral) Chapter 2 Problem 14 You have three new amino acids, x, y, and z, with the following structures: These are joined to make a tripetide with the sequence N—x—y—z—C. Calculate the average charge on the tripeptide to two decimal places at 9.0 of x y z.  Chapter 2 Problem 14 You have three new amino acids, x, y, and z, with the following structures: These are joined to make a tripetide with the sequence N—x—y—z—C. Calculate the average charge on the tripeptide to two decimal places at 9.0 of x y z. Charge at pH of 9.0 C-term (pKa 3.0) YR1 (COOH pKa 4.4) YR2 (COOH pKa 5.2 N-term (pKa 10.1) xR (OH pKa 10.3) NET  Chapter 2, Problem 11b b) Pepe supplied you with this charge vs. pH titration curve for his peptide. Pepe also told you that the N-terminal residue contains sulfur, and that the side chain of the C-terminal residue possesses a 6-membered ring. Indicate the residue sequence of this peptide by placing one amino acid in each of the blanks below. (There is more than one correct answer.) N -- ____________ -- ____________ -- ____________ -- ____________ -- C  Key Functional Groups to Know and be able to Apply Acid Form [HA] Base form [A-] Example Structure Charge Structure Charge −NH3+ +1 −NH2 0 ɑ-amino R-group of Lys =NH2+ +1 =NH 0 R-group of Arginine ≡NH+ +1 ≡N 0 R group of His COOH 0 COO- -1 ɑ- COOH R-groups of Asp and Glu OH 0 O- -1 R-group of Tyrosine SH 0 S- -1 R-group of Cysteine Without looking them up, see if you can name the triprotic amino acid whose R group is shown! Course Business Before lab this week you will need to upload a free educational version of PyMOL onto your computer. Please look at the prelab early What if you were asked about a section in the middle of a peptide Which end is the amino end? O Left A. B. Right  Module 1-4 Question 2 c. If the second residue were replaced with arginine would the pI increase or decrease? Answer the question by calculating the pI of the old and new polypeptide and pKa 9.7 compare them. pKa 2.1 Can we make it make sense conceptually? pKa 6.0 Valine-Isoleucine-Histidine- Threonine Valine-Arginine-Histidine- Threonine  +2 + 1 - 1 2. 6 0. 9. 7 - EN NH2 100 100 H ENH + NHst 6 0+ 9 7.. = 7 83. 2 A. pl ↑ B. pIt ① I 0" + 3 +2 - 6 a 7 12. 2 1... NHz NHz 100 - EN + NHaT NHat 100 H ENH 911. First Approach: Consider it Conceptually The Rule of Thumb pI increases when the side chain’s charge increases. Any of the following: 0 + -+ -0 pI decreases when the side chain’s charge decreases. Any of the following: 0 - +- +0 *Non-ionizable side chains are neutral (0) Isoelectric Point (pI) and solubility Effect of pH on milk protein (casein) Maureen Daschel: https://www.youtube.com/watch?v=egazCtStwsQ The solubility of the amino acid or protein is the lowest at its pI  Isoelectric Point (pI) of a Polypeptide- Practical Use Isoelectric Focusing: Separates proteins by their isoelectric point! solution of molecules applied to the gel. More positive molecules below their pI, attracts cations attracts anions more negative above the pI Anions drawn to anode; cations drawn to cathode eventually stall when they hit their pI (neutral)  Protein Structure A B C D Primary Secondary Tertiary Quaternary secondary structure is stabilized by : E: all levels are found A. H-bonds blu atoms Of backbone in all proteins to multiple ? only applies Which level of structure is only found in some proteins? Learning Objectives You should be able to… Regarding Primary Structure In addition to labeling the peptide bond, you should be able to label the phi and psi bonds explain which bonds in a polypeptide have free rotations and which ones are rigid. Regarding Secondary Structure (Part I) identify and explain how the groups on a polypeptide participate in formation of secondary structure for both alpha helices and beta pleated sheets. regarding alpha helices specifically, recall how many residues/turn, how many degrees between R groups, the handed-ness of the helix, the n+4 rule, and where hydrogen bonds are occur regarding beta-pleated sheets, differentiate between parallel and anti-parallel sheets and explain the reason for the pleated appearance Below is the primary structure of a protein Module Quiz 3-1 Question 1 a. For each part of the question, select the letter associated the with appropriate component of the structure. Peptide bond: _____ Psi bond: Phi bond: N-terminus: i _____ _____ _____ C-terminus: _____ A b. Which letter represents a bond without 360-degree rotation? _____ B The peptide bond is rigid, due to its partial double character. A protein’s three-dimensional shape comes from rotation around its phi ɸ and psi Ѱ bonds. Theoretically, there is a 360o rotation around each of these bond. The peptide bond is rigid, due to its partial double character. A protein’s three-dimensional shape comes from rotation around its phi ɸ and psi Ѱ bonds. Theoretically, there is a 360o rotation around each of these bond. Is the conformation: A. Cis B. Trans O  The most common conformation for peptides is the “trans” configuration positions the R groups away from one another dipeptide Steric Hinderance: when atoms/molecules try to take up the same space it cause electron clouds to overlap, which causes a repulsive force and influences which bond butane Steric Hinderance! angles are more likely to occur +1800 00 Side view anti Staggered “ ” 0 to +180o for angles that make with a clockwise rotation; 0 to -180o for angles that make with a counter-clockwise rotation The most common conformation for peptides is the “trans” configuration positions the R groups away from one another R H O R Cα N C Cα C Cα N O R H Theoretical Rotation around Phi (ϕ) and Psi (ψ) R H O R Cα N C Cα C Cα N O R H Phi ϕ Psi ψ a to sen R R R  Theoretical Rotation around Phi (ϕ) and Psi (ψ) R H O R Cα N C Cα C Cα N O R H Phi ϕ 0o +90o +/-180o -90o phi ɸ: Right-handed rotation around Ca-N imagine the C=O being the hand that sweeps around the clock A range of angles are favored depending on the residue’s R group and neighboring residues. Certain angles are not favored due to steric hinderance. This gif shows the rotation of the phi bond while psi is held constant  Theoretical Rotation around Phi and Psi 0o +90o +/-180o -90o psi Ѱ: Right-handed rotation around Ca-C imagine the N being the hand that sweeps around the clock  Ramachandran Plot shows the Phi and Psi Angles that are Sterically Allowed in the Backbone Phi ϕ : 80o Psi ψ : 45o A. α-helix (right-handed) O B. α-helix (left-handed) C. β pleated sheet (parallel) Gopalasamudram Narayanan D. β pleated sheet (anti) 450- · Ramachandran White Space: Angles not allowed Blue Regions: Allowed Conformations for phi/psi. Specific phi/psi angle leads to Alpha Helices and jo Beta-Pleated Sheets. alpha helices: φ [phi] = –60 deg; ψ [psi] = –45 to –50 deg beta sheets: φ [phi] = -140 deg ; ψ [psi] = +130 deg. Within those ranges (where Rs are pointed away), Secondary Structures forms as a Result of a Polypeptide Maximizing the Number of H-bonds it can make between its carbonyls and amides from its backbone These are the most stable conformations for portions of polypeptides to assume Learning Objectives You should be able to… Regarding Primary Structure identify the L versus D enantiomer if given a wedge and line drawing of an amino acid In addition to labeling the peptide bond, you should be able to label the phi and psi bonds explain which bonds in a polypeptide have free rotations and which ones are rigid. Regarding Secondary Structure (Part I) identify and explain how the groups on a polypeptide participate in formation of secondary structure for both alpha helices and beta pleated sheets. regarding alpha helices specifically, recall how many residues/turn, how many degrees between R groups, the handed-ness of the helix, the n+4 rule, and where hydrogen bonds are occur regarding beta-pleated sheets, differentiate between parallel and anti-parallel sheets and explain the reason for the pleated appearance Module Quiz 3-1 Question 2: Imagine that the peptide below is an alpha helix. A. First, determine is the N-terminus on the left or right? B. Determine the two atoms that participate in the one hydrogen bond responsible for its alpha helical secondary structure. The atoms are labeled 1-13 in the image below. Select the lowest number first, followed by the higher number. lowest number: ______ 3 highest number: _______ 13 C. The atom that donate a H-bond comes from the __________ Nitrogen of The atom that accepts a H-bonds comes from the an _________ of oxygen carbony ( a amide ]DDD : H-bonds between carbonyls and amides of the backbone stabilize the helix The carbonyl of the earlier residue hydrogen bonds with the amide of the later residue, 4 away (n+4 rule). 3.6 residues/ term All carbonyl oxygens point towards the Carboxyl term side of helix All amide nitrogens point towards the Amino term side of helix NO, groups pointing away fro