Week 2: Amino Acid and Protein Structure

peptides: a variety of function

smaller strings of amino acids and are likely modified • Hormones and pheromones - insulin (think sugar metabolism) - oxytocin (think childbirth) - sex-peptide (think fruit fly mating) • Neuropeptides - substance P (pain mediator) • Antibiotics - polymyxin B (for Gram - bacteria) - bacitracin (for Gram + bacteria) • Protection, e.g., toxins - amanitin (mushrooms) - conotoxin (cone snails) - chlorotoxin (scorpions)

Glutamate (-) titration curve

Aspartate curve will look similar

Amino acids without ionizable R-groups can act as a zwitterion in a(n) _____ solution. nonpolar boiling acidic basic neutral

neutral

Asparagine, Asn, N

Polar, uncharged R groups

cysteine - cys - C

Polar, uncharged R groups

glutamine - gln - Q

Polar, uncharged R groups

serine - ser - S

Polar, uncharged R groups

threonine - thr - T

Polar, uncharged R groups

Almost all residues in polypeptides are in trans configuration

The trans form is strongly favored because of steric clashes, indicated by the orange semicircles, that arise in the cis form. Proline is an exception to this rule:

Protein Purification: Chromatographic methods

The workhorse of modern biochemistry Charge - Ion exchange chromatography Size (Molecular Mass)- Size exclusion chromatography Cofactors - Affinity chromatography Hydrophobicity - hydrophobic chromatography, (NH3)2SO4 Precipitation ("Salting out")

1. Separation by charge: ion exchange chromatography

This is cation exchange Chromatography Cations bind Other cations are used to complete with the bound ones Resin will have negative charge positive charge will be retained longest net negative charge will move quickly Cation exchange: Proteins with pI higher than the pH of buffer are positively charged and bind to the column material. Positively charged proteins stick to negative beads. -Proteins with pI lower than the pH of buffer are negatively charged and flow through the column -Elution of the bound proteins: Use increasing salt concentration (common) or change pH (much less common - why: pH is harder to change?) cations in salt will also bind to resin and at high concentrations will compete with proteins and separate from resin so proteins would flow through How would anion exchange work? Reverse charges and concepts

proline isomers

Trans and cis X-Pro bonds. The energies of these forms aresimilar to one another becausesteric clashes, indicated by theorange semicircles, arise in Ca both forms. Proline Isomers Most peptide bonds not involving proline are in the trans configuration (>99.95%) For peptide bonds involving proline, about 6% are in the cis configuration. Most of this 6% involve β-turns Proline isomerization is catalyzed by proline isomerases

2. Lyse the cells and make the protein happy (pH, temp., ionic strength)

Use a reasonable buffer system • What make a "happy" protein? - Proper pH (usually neutral) -Choose buffer with pKa near chosen pH • Reasonable salt concentration -Typically ~150 mM Na/K Cl • Temperature -Usually work at 4° C, or on ice • Cofactors/ligands -Many proteins are more stable with all necessary components • Cryoprotectant -Proteins stored frozen frequently include glycerol or other cryoprotectants. • Detergents - Membrane associated proteins will need detergents to dissociate and stabilize protein.

Size exclusion chromatography plot

Ve = elution volume of protein Relative elution volume actually depends on hydrodynamic radius Rs, which is proportional to mass - assuming spherical shape. B: volume x and log size of protein x

Elution volume vs Void Volume

Ve: elution volume, will be smaller for larger proteins bc small proteins elute first

Common Questions About Peptides and Proteins

What is its sequence and composition? (sequence gene that encodes protein) What is its three-dimensional structure? (final structure and folding) How does it find its native fold? How does it achieve its biochemical role? (structure of protein can shed insight to role) How is its function regulated? (regulated at the expression level and modified, upregulate or down regulate, or binding from another molecule) How does it interact with other macromolecules? How is it related to other proteins? Where is it localized within the cell? What are its physico-chemical properties? (size, charge, ligameric state)

Protein Purification Methods Questions

What is the method used for? How is the method accomplished (a couple sentences) What kind of results do we get? How are the results analyzed?

Purify the Following Protein: he ATPase DnaK is a 68 kDa protein with a pI of 4.6. How would you get this protein pure?

3 techniques to use:

Selective precipitation of a protein from a crude extract is MOST effective by which molecule? ammonium sulfate urea sodium dodecyl sulfate EDTA (ethylenediaminetetraacetic acid) All of the answers are correct.

ammonium sulfate

Negatively charged R groups

aspartate, glutamate Also called "Aspartic acid" and "Glutamic acid". Note similarity to asparagine and glutamine.

valine - val - V

non-polar, aliphatic

Two Cys residues can be joined by a disulfide bond

oxidation reaction Disulfide bonds can stabilize a protein or join two chains together.

glutamic acid - glu - E

Acidic, negatively charged (hydrophilic)

Glycine (0 net charge) Titration curve

*All other uncharged AA curves will look similar serine, threonine, cysteine, asparagine, glutamine

Separation relies on differences in physical and chemical properties like...

- charge - size - affinity for a ligand - solubility - hydrophobicity - thermal stability Chromatography is commonly used for preparative separation in which the protein is often able to remain fully folded.

Aspartic acid, Asp, D

Acidic, negatively charged R group, (hydrophilic)

protein structure and polypeptide size

Amino acids - the building blocks Amino acid structure and classification Proteins are built from a linear "string of amino acids Protein structure - Primary: sequence - Secondary - Tertiary - Quaternary Polypeptide Size and Number Varies Greatly in Proteins

Amino acid charges

Amino acids are zwitterions (ampholytes) at neutral pH pH < pKa: protonated pH > pKa: deprotonated

Amino acids can act as buffers

Amino acids with uncharged side chains, such as glycine, have two pKa values: The pKa of the a-carboxyl group is 2.34 The pKa of the a-amino group is 9.6 It can act as a buffer in two pH regimes.

Lysine (+) titration curve

Arginine curve will look similar

tryptophan - trp - W

Aromatic R groups

tyrosine - tyr - Y

Aromatic R groups

Estimating the pI of a Protein (Polypeptide)

Basic protein: # of K, R, H >> D, E (+) at pH 7 pI > 7 (high) Acidic protein: # of D, E >> K, R, H (-) at pH 7 pI < 7 (low) *Protein charge depends on pH* (in addition to types and amounts of acidic/basic side chains)

Separate soluble fraction from insoluble fractions

Cellular fractions can be separated by differential centrifugation. We may jump right to last step for protein purification. Proteins can be purified according to solubility, size, charge, and binding affinity

3. Separation by Unique Binding: Affinity chromatography

Covalently couple/ ligand mimics and protein will stick to beads. Change solution containing excess ligand and protein will bind and elute protein bound to ligand We can engineer an affinity tag onto recombinant proteins. - recombinant engineering to add extra amino acids The most common is 6-His, which binds nickel (and some other metals) with high affinity. - binds protein to nickel and all proteins that do not bind will elute - A resin with bound nickel is used as stationary phase.Protein is eluted with imidazole (why?).

Exploit Physical Properties Among Proteins - Density

Creation of a density gradient can be used to separate biomolecules. more dense at bottom to separate sample

Isoelectric point for a polypeptide (protein)

Determined by the number of charged amino acids Protein with large number of D, E: low pI negatively charged at physiological pH Protein with large number of K, R, H: high pI positively charged at physiological pH

Favorable Interactions in Proteins

Hydrophobic effect- The release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy. Hydrogen bonds- Interaction of N−H and C=O of the peptide bond leads to local regular structures such as a helices and b sheets. London dispersion - Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein. Electrostatic interactions - long-range strong interactions between permanently charged groups - Salt bridges, especially those buried in the hydrophobic environment, strongly stabilize the protein.

3 Main Types of AA Titration Curves

In general (when at pH 7 at physiological conditions): 1.Net zero charge (Glycine and other uncharged AAs) 2.Negatively charged, acidic (Glutamate, Aspartate) 3.Positively charged, basic (Lysine, Arginine) *Histidine technically follows the pattern of #3... However, the graph looks a bit different than that of Lys/Arg because pK1, pKR, and pK2 are equidistant (more symmetrical)

Simple (no charged R-groups) amino acids as buffers

Isoelectric point pI: pH at which net charge is 0 pI=pK1 +pK2 /2

Chromatography

Methods that separatemolecules by exploitingphysical and chemicalproperties Note: Stationary phase (solid phase) Mobile phase: our solution that contains protein then goes to stationary phase with specific chemical properties to separate detection device at the end measures UV absorbance

Amino acids with charged R-groups: 3 transitions

Neg. charged R: pI = 1/2 (pK1 + pKR) pI = 3.22 Pos. charged R:pI = 1/2 (pKR + pK2)

phenylalanine - phe - F

Non-polar aliphatic aromatic

Alanine, Ala, A

Nonpolar, aliphatic R Groups

glycine - gly - G

Nonpolar, aliphatic R Groups

isoleucine - ile - I

Nonpolar, aliphatic R Groups

leucine - leu - L

Nonpolar, aliphatic R Groups

methionine - met - M

Nonpolar, aliphatic R Groups

proline - pro - P

Nonpolar, aliphatic R Groups

Uncommon Amino Acids in Proteins

Not incorporated by ribosomes - except for Selenocysteine Arise by post-translational modifications of proteins Reversible modifications, especially phosphorylation, are important in regulation and signaling

Polypeptides: ionizable pKr values contribute to the total charge

Note: One terminal amino group, one terminal carboxyl group, multiple titratable side chain groups per polypeptide.

Peptide Ends Are Not the Same

Numbering (and naming) starts from the amino terminus (N-terminal). AA1 AA2 AA3 AA4 AA5 Using full amino acid names: serylglycyltyrosylalanylleucine Using the three-letter code abbreviation: Ser-Gly-Tyr-Ala-Leu For longer peptides (like proteins) the one- letter code can be used: - SGYAL

Amino acid chirality

Only the L isomer of amino acids are found in proteins. Amino acids differ at the R position. All amino acids are chiral (except glycine) Proteins only contain L amino acids • The α carbon always has four substituents and is tetrahedral. •All (except proline) have: - an acidic carboxyl group connected to the α carbon - a basic amino group connected to the α carbon - an α hydrogen connected to the α carbon • The fourth substituent (R) is unique to each amino acid. In glycine, the simplest amino acid, the fourth substituent is also hydrogen.

Amino acid: atom naming

Organic nomenclature: start from one end Biochemical designation: - start from a-carbon and go down the R-group

histidine - his - H

Polar basic, positive charge

lysine - lys - K

Polar basic, positive charge

Arginine, Arg, R

Polar basic, positive charge r group

Studying Proteins and Peptides Sometimes Requires Purification from a Mixture

Polypeptides contain differing amino acid sequences. The sequence and arrangement of amino acids gives the polypeptide a chemical character (i.e., charged, polar, hydrophobic, etc.). Some polypeptides bind specific targets, which can be used to "fish them out" of a complex mixture.

Protein Structure

Rotation around bonds in peptides leads to unlimited potential folding Each protein has a specific function Implies that each amino acid sequence attains only 1 favored three-dimensional form Native conformation: allows to perform designated function Implies that proteins have only 1 or a few thermodynamically stable Native Conformations Also implies that structure leads to function Altering even one amino acid can inhibit function Examples: Sickle Cell Anemia and Cystic Fibrosis Unlike most organic polymers, protein molecules adopt a specific three-dimensional conformation. This structure is able to fulfill a specific biological function. This structure is called the native fold. Unfolded proteins are referred to as denatured. The native fold has a large number of favorable interactions within the protein. There is an entropy cost to folding the protein into one specific native fold.

Common resins

S cation exchange or CM Negatively charged groups bind to positively charged ligands. Anion exchange

Protein purification steps

STEP 1: Pick a protein source -Isolate protein from organism in which it is abundant -Isolate protein from tissue in which it is abundant -Overexpress protein in E. coli or other expression system • Requires recombinant DNA technology (Chapter 9) - sources: livers, large animals, other animals Recombinant protein sources Many organisms can be engineered to produced a protein of interest: -E. coli bacteria -Yeast -Insect cells -Mammalian cells -Plants: Green factory: Plants as bioproduction platforms for recombinant proteins. These are done in fermentation (tissue culture) conditions in the lab environment. After picking a protein source... 1. Grow the cells (e.gE.coli) and make a lot of the desired protein 2. Lyse the cells and make the protein happy: Lyse the cells and make the protein happy (pH, temp., salt) • Freeze/thaw, enzymes (lysozyme), and/or... -Waring blender -French press -Sonicator (old school) (pH, temp., salt) 3. Purify the protein

Step 2: Exploit Physical Properties Among Proteins - Solubility

Salting out Salting out takes advantage of the fact that the solubility of proteins varies with the salt concentration. Most proteins require some salt to dissolve in water, a process called salting in. As the salt concentration is increased, different proteins will precipitate at different salt concentrations, a process called salting out. Solubility -Salting out of proteins (Ammonium Sulfate) -Disrupt "hydrophobic patches" on surface of proteins -Proteins precipitate (hopefully without denaturation) and are separated by centrifuge. Recombinant thermophilic proteins can be separated by heating sample which denatures and precipitates most proteins native to host organism. We can alter the solubility of proteins by increasing salt concentration. We can then fractionate by precipitation.

elution volume

Separation by size: size exclusion chromatography Ve = elution volume of proteinVo = void volume (exclusion volume) of column Relative elution volume Ve/Vo depends on molecular mass(assuming spherical shape) -shortest path from pump to end Larger molecules elutes sooner than smaller molecules as they have less volume/distance to travel so lowest Ve Ve = elution volume of protein Relative elution volume Ve/Vo actually depends on hydrodynamic radius, which is proportional to mass - assuming spherical shape. -plots elution volume vs size

Primary Structure: Amino acids are linked by peptide bonds to form polypeptide chains

The polypeptide consists of a repeating part called the main chain or backbone and a variable part consisting of the distinctive amino acid side chains. The backbone has hydrogen bonding potential because of the carbonyl groups and hydrogen atoms that are bonded to the nitrogen of the amine group. Most proteins consist of 50 to 2000 amino acids. The mean molecular weight for an amino acid is 110 g mol-1. Protein mass can also be referred to as Daltons.

Dialysis

The salt can be removed from a protein solution by dialysis. The protein solution is placed in a cellophane bag with pores too small to allow the protein to diffuse, but big enough to allow the salt to equilibrate with the solution surround the dialysis bag. changes buffer systems

Zwitterion, pI, and pKa

Zwitterion = amino acid when its net charge is 0 pI = pH at which amino acid is in zwitterion form (net charge of 0) (^stands for isoelectric point) Amino acids don't just have a single pKa... ●pKa for the carboxyl group being deprotonated = pK1 ●pKa for the amino group being deprotonated = pK2 ●pKa for ionizable R group(s) being deprotonated = pKR#

In the diagram below, the plane drawn behind the peptide bond indicates the: a. absence of rotation around the C—N bond because of its partial double-bond character. b. plane of rotation around the Cα—N bond. c. region of steric hindrance determined by the large C=O group. d. region of the peptide bond that contributes to a Ramachandran plot. e. theoretical space between -180 and +180 degrees that can be occupied by the φ and ψ angles in the peptide bond.

absence of rotation around the C—N bond because of its partial double-bond character.

Step 2: Exploit Physical Properties Among Proteins

after choosing protein source and buffer chromatography: technique to separate proteins based on their common properties such as: Size: Size exclusion chromatography -length of polypeptide Charge: Ion exchange chromatography Cofactors: Affinity chromatography -Properties like charge can be identified with Io -protein of interest has an affinity for column Hydrophobicity: hydrophobic chromatography -Hydropathy index E. coli~ 3000 different proteins Humans > 30,000

The functional differences, as well as differences in three-dimensional structures, between two different enzymes from E. coli result directly from their different: affinities for ATP. amino acid sequences. roles in DNA metabolism. roles in the metabolism of E. coli. secondary structures.

amino acid sequences.

Which factor is LEAST likely to result in protein denaturation? altering net charge by changing pH changing the salt concentration disruption of weak interactions by boiling exposure to detergents mixing with organic solvents such as acetone

changing the salt concentration

Nonpolar, aliphatic R groups

glycine, alanine, proline, valine, leucine, isoleucine, methionine

4. Hydrophobic Interaction Chromatography

hydrophobic regions will associate together and will form aggregates in clusters, hopefully won't be natured or unfolded decrease salt decreases affinity of protein for column Works best with amphipathic proteins and small proteins Polar and non-polar regions Exploits non-polar regions of proteins in high salt conditions Causes them to bind to each other and aggregate proteins Similar in concept to"Salting Out" Steps: 1.Add the protein mixture to the column in a solvent with a high concentration of salt Ammonium sulfate Sodium chloride Lithium chloride 2. Proteins lose their solvation layer of water and exposes hydrophobic patches 3. Hydrophobic patches bind to the column support 4. Protein is elute from the column using adecreasing salt gradient

Ion exchange b size exclusion: Sephadex, a Dextran

ion exchange: smooth smooth beads Basis of size extraction larger proteins cannot go as far in beads and will be excluded, so it would have smaller volume in interior of beads

The chirality of an amino acid results from the fact that its α carbon: has no net charge. is a carboxylic acid. is bonded to four different chemical groups. is in the L absolute configuration in naturally occurring proteins. is symmetric.

is bonded to four different chemical groups.

Positively charged R groups

lysine, arginine, histidine pKas will change in catalytic mechanisms

Separation by size: size exclusion chromatography

measures size of quaternary structure Long narrow columns isocratic solution larger proteins would get excluded

Reversible Modifications of Amino Acids

most common: phosphorylations transferred or removed

Aromatic R groups

phenylalanine, tyrosine, tryptophan ring with conjugate double bond These amino acid side chains absorb UV light at 270-280 nm

Modified Amino Acids Found in Proteins

protein maturation permanent modifications! More than 250 non-standard amino acids. Most are derivatives of the 20 common ones.

Resonance in the Peptide Bond

resonance allows for redistribution of electrons resulting in changes in rotations due to double bond characteristics (planar) The polypeptide is made up of a series of planes linked at α carbons The Rigid Peptide Plane and the Partially Free Rotations -Rotation around the peptide bond is not permitted -Rotation around bonds connected to the alpha carbon is permitted: BUT rotation is allowed around the single bond between: ●alpha C and amide-N (phi Φ) ●alpha C and carbonyl-C (psi Ψ) f (phi): angle around the a-carbon—amide nitrogen bond y (psi): angle around the a-carbon—carbonyl carbon bond In a fully extended polypeptide, both y and f are 180°

Polar, uncharged R groups

serine, threonine, cysteine, asparagine, glutamine These amino acids side chains can form hydrogen bonds. Cysteine can form disulfide bonds.

Protein Stability and Folding

• A protein's function depends on its 3D structure. • Loss of structural integrity with accompanying loss of activity is called denaturation. • Proteins can be denatured by: heat or cold pH extremes organic solvents chaotropic agents (urea and guanidinium hydrochloride)

Proteins are made from 20 common amino acids

• Common amino acids can be placed in five basic groups depending on their R substituents: Nonpolar, aliphatic (7) Aromatic (3) Polar, uncharged (5) Positively charged (3) Negatively charged (2) All amino acids can be referred to by one- and three- letter codes.

How to Calculate the pI When the Side Chain Is Ionizable

• Identify species that carries a net zero charge. Identify the pKa value that defines the acid strength of this zwitterion: (pKR). Identify the pKa value that defines the base strength of this zwitterion: (pK2). Take the average of these two pKa values. What is the pI of histidine?

Formation of Peptides

• Peptides are small condensation products of amino acids • They are "small" compared to proteins (Mw < 10 kDa) Note: Loss of H2O in condensation reaction. carboxyl group to carbonyl to form peptide bond

Proteins are composed of:

• Polypeptides (covalently linked a-amino acids) + possibly: • cofactors - functional non-amino acid component - metal ions or organic molecules • coenzymes - organic cofactors - e.g. NAD+ in lactate dehydrogenase • prosthetic groups- covalently attached cofactors (flavin) - heme in myoglobin • other modifications (posttranslational modifications)

Amino Acids: Building Blocks of Protein

• Proteins are linear heteropolymers of a-amino acids linked in covalent peptide bonds • Amino acids have properties that are well-suited to carry out a variety of biological functions - Capacity to polymerize - Useful acid-base properties - Varied physical properties - Varied chemical functionality

Working with proteins: purification and analysis

• Purifying proteins - Source of protein - Separation • Salting out • Chromatography • Analysis - How much protein do I have? - Is my protein pure? - What components do I have? - What is the structure?

Proteins: Agents of biological function

•Catalysis - Hexokinase (Glycolysis) - DNA polymerase (DNA replication) • Nutrient Transport - Hemoglobin (O2 transport in the blood) - Glucose transporter (Glucose transports across the cell membrane) • Structure - Collagen (connective tissue) - Keratin (hair, nails, feathers, horns) - Cytoskeleton (e.g. intermediate filaments, micro tubules) (muscle) (muscle) • Motility - Myosin (muscle) - Actin (muscle)