BIOC 431 (Dr. Barycki) Midterm 1 Study Guide

Attraction or repulsion due to charges.

      Highlight = Equations  Highlight = Important Concepts  Highlight = Key Terms

Chapters 2 and 3 ­ Water and Amino Acids, Peptides, and Proteins (Week 1)

Important Basic Terms to Know 

­ Hydrogen bonds: Dipole­dipole or charge­dipole interactions occurring between two electronegative atoms (a proton acceptor and a proton donor)

­ Typically 4­6 kj/mol in strength (weak)

      ­ Ionic Interactions: Attraction or repulsion due to charges

­ Hydrophilic Compound: A compound that dissolves in water (water­loving). ­ Polar or charged compounds

­ Hydrophobic Compound: A compound that does NOT dissolve in water (water­hating). ­ Nonpolar compounds (i.e. lipids)

­ Amphipathic Compound: A compound that has both hydrophobic and hydrophilic properties.

­ Phospholipid bilayer in cell membranes, soap, etc.

What are the three main ways to separate proteins?

­ Hydrophobic Interactions: Forces that cause nonpolar regions of molecules to cluster together.

­ Van der Waals Interactions: Weak attractive forces between any two uncharged atoms.

­ Buffer: Resists drastic changes in pH by converting strong acids or bases into weak ones. If you want to learn more check out What is a linguistic?

­ Bicarbonate, etc.

­ Buffering Region: This is the flat zone on a titration curve where there is the strongest resistance to a change in pH.We also discuss several other topics like What is Regency?

­ Amino Acid Residue: An amino acid linked to another via a peptide bond in a polypeptide chain.

­ PI: This is the isoelectric point or isoelectric pH, which is the pH where there is no net electric charge of an amino acid

Water 

­ The solubility of a molecule is mainly determined by its interactions with water ­ If a molecule is polar or charged, it will dissolve in water We also discuss several other topics like what is James Lange theory?

­ If a molecule is predominantly hydrophobic, due to its nonpolar character, it will cluster together with other hydrophobic molecules (via hydrophobic interactions) in order to have the most favorable entropy (randomness) by causing the water molecules around to be less ordered around the cluster.

Tendency of a protein to maintain its native conformation.

­ The Henderson­Hasselbalch equation can be used to describe the equilibrium of weak acid deprotonation 

­ pH = pKa + log ([A­]/[HA]) 

­ 10(pH­pKa) = [A­]/[HA] 

­ When there are equal concentrations of A­ and HA, the pka = the pH ­ pKa = ­log (Ka) 

­ pH = ­log ([H+]) 

­ Buffers work best 1 pH unit above or below the pka We also discuss several other topics like what is Greenwashing?

Amino Acids and Peptides 

­ Amino Acids are the building blocks of proteins and consist of 4 parts:       1. An alpha carbon, which is a chiral center (except in glycine)

      2. An alpha amino group 

      3. A carboxylic acid 

      4. A side chain (R­group) 

­ Amino acids bind to each other to create polypeptides through covalent bonds called Peptide Bonds 

­ These occur between the alpha carboxyl group of one amino acid and the alpha amino group of another 

­ This happens via a dehydration reaction.

­ In a polypeptide chain, the N­terminus is at the beginning and the C­terminus is at the end.

­ Amino Acids are Zwitterionic (both (+) and (­) charged) at neutral pH ­ Amino group gives the positive charge

­ Carboxylic acid group gives the negative charge

­ **Some charged R­groups can further influence overall charge of amino acids**

      ­      ** Amino Acids with R­groups that are charged**:

       ­     Negative→ Aspartate (Asp) + Glutamate (Glu)

       ­     Positive→ Histidine (His), Lysine (Lys), and Arginine (Arg)

­ The pKa of a carboxylic group is ~2.1 and the pKa of an amino group is ~9.5 

­ Aromatic Amino Acids Absorb UV light 

­ Tryptophan (Trp), Tyrosine (Tyr), and Phenylalanine (Phe) all absorb UV light, with Trp being the best (due to its abundant conjugation of pi electrons), then Tyr (due to its resonance with the extra oxygen), and then Phe.

Working With Proteins 

­ Proteins are able to be separated by 3 different physical properties: 

­ Size If you want to learn more check out What is the inability to get everything we want due to a finite amount of resources?

­ Charge

­ Function

­ What is a pI? Don't forget about the age old question of What is a self schemas?

­ A pI is the pH where an amino acid has no net charge.

­ A pI can NOT be calculated. It must be determined experimentally.

­ If an amino acid has a pI of 8, then at a pH of 6 (below the pI), that amino acid would most likely have a positive charge (not always though).

­ At a pH of 10 (above the pI), that amino acid would most likely have a negative charge (not always though).

­ Therefore, generally this is true: ←+​ pI­​→

→ This illustrates the general rule that a pH above the pI makes the 

amino acid negative, and a pH below the pI makes the amino acid 

positive.

­ There are 3 main ways to separate proteins: 

­ Ion Exchange Chromatography

­ Size Exclusion Chromatography

­ Affinity Chromatography

­ Ion Exchange Chromatography: 

­ In this method of protein separation, a column full of beads with a positive or negative charge are used to separate proteins based on their different charges.

­ Cation Exchange Columns have negatively charged beads that are intended to slow positively charged proteins down (due to attraction), while negatively charged proteins elute much quicker due to repulsion.

­ Anion Exchange Columns have positively charged beads that slow down negatively charged proteins (due to attraction), while positively charged proteins elute much quicker due to repulsion.

­ To elute the proteins stuck in the beads due to charge, you can either salt the column or change the pH of the column. Salting it neutralizes all of the charges, causing the proteins to release from the beads. Changing the pH changes the charge of the proteins, causing them to release from the beads.

­ Size Exclusion Chromatography: 

­  This method of separation uses small cavities in the resin of the column to separate proteins by their size. Pressure is the force of separation.

­ The small cavities cause the small and medium sized proteins to get more “caught up” in the cavities.

­ The larger proteins will go around the cavities, eluting through the column much quicker. This is due to the fact that they don’t fit into the cavities.

­ Although this is a good method of protein separation, it cannot separate proteins of similar molecular weight (MW). They would elute at around the same time.

­ Affinity Chromatography: 

­ This separation method uses specific ligands covalently attached to the beads of the column that are specific to the function of the target proteins.

­ The target proteins bind to the ligands and are slowed down, while contaminants are eluted through first.

­ For Purification of Recombinant Proteins: 

­ Immobilized Metal Affinity Chromatography 

­ Recombinant proteins can be tagged with (His)6tags (bind to Ni2+) or Glutathione­S­transferases (GST ­ binds to glutathione).

­ Depending on the type of tag the protein has, the beads of the column will contain the substance the tags have affinities for.

­ When the target protein passes through the column, it will stick to the beads due to its affinity for them.

­ To elute the target proteins, you can change the pH to alter the proteins’ charges or add imidazole to the column.

­ How do you determine the purity, oligomeric state, or MW of your protein? → → Polyacrylamide Gel Electrophoresis (PAGE) ← ←

­ Polyacrylamide Gel Electrophoresis (PAGE) 

­ This method of visualization is based on the migration of proteins in an electric field based on their sizes. 

­ This is NOT a way to separate proteins efficiently!! Too small of an amount. ­ This technique allows one to know the number of different proteins in mixtures, the molecular weight of proteins, the purity of a protein, or the isoelectric point of a protein.

­ The polyacrylamide gel has negative and positive electrodes at opposite ends, and it is made up of a polymer that causes bigger proteins to get caught up in the structure. Therefore, the smaller the proteins will move faster along the gel.

­ There are 4 types of PAGE: 

­ Reducing SDS­PAGE

­ Non­reducing SDS­PAGE

­ Native PAGE

­ Isoelectric focusing PAGE

­ Reducing SDS­PAGE 

­ This “reduces” proteins by using DTT or BME to break disulfide bonds, and it also uses SDS which denatures the proteins and gives them all negative charges so they move by MW down towards the positive cathode.

­ Non­reducing SDS­PAGE 

­ This does not reduce disulfide bonds, but when next to a Reducing SDS­PAGE, it can be used to determine whether there are intersubunit disulfide bonds in a protein.

­ Native​ ​PAGE 

­ This is PAGE of a protein in its native conformation, and it is done without SDS or Reducing agents. When next to the others, it can help determine the oligomeric state of a protein. 

­ Isoelectric Focusing PAGE 

­ This is used to experimentally determine the isoelectric point of proteins in their native conformations. This is NOT a protein separator. Purely diagnostic. ­ This works by creating a pH gradient with a solution, and when the proteins are placed on top, they will stop moving when they have no net charge. This pH marker where they stop is their isoelectric pH or isoelectric point.

Chapter 4 ­ The Three­Dimensional Structure of Proteins (Week 2 + 3) Protein Structure: Primary, Secondary, Tertiary 

­ Primary Structure 

­ The amino acid sequence of a protein

­ Secondary Structure 

­ The spatial arrangement of the polypeptide chain’s atoms

­ Involves alpha helices, beta sheets, beta turns, etc

      ­    ​Tertiary Structure 

­ The 3D arrangement of all atoms in proteins

­ Combines 2 or more secondary structures → Known as Motifs 

­ Examples are Beta­Barrels, Beta­Alpha­Beta Loops, etc.

­ Proteins in their functional, folded conformations are called Native Proteins 

­ Stability is the tendency of a protein to maintain its native conformation ­ Free energy impacts the conformation of a protein 

­ 3 principles determine the conformation:

    1. “Like dissolves like” 

     2. Must be a lower free energy for a protein to fold than not to fold 

      3. Two atoms can NOT be in the same place at the same time 

­ 4 non­covalent interactions stabilize protein structures: 

­ Hydrogen Bonds

­ Hydrophobic Interactions

­ Electrostatic Interactions

­ Van der Waals Interactions

­ Hydrogen Bonds 

­ Play a big part in structure whether it’s Water­Water, Water­Protein, or Protein­Protein.

­ Hydrophobic Interactions/Clustering 

­  Plays an important role in structure through clustering when in the presence of water in order to increase entropy (decrease order of water molecules around). ­ For this reason, most proteins have a hydrophobic core, containing nonpolar amino acid residues in the core. This way entropy is favorable.

­ Electrostatic Interactions/Charge Neutralization 

­ These interactions include salt bridges or ionic interactions where 2 opposite charges attract each other

­ These interactions are the strongest when they are in a hydrophobic environment where there is a low dielectric constant.

­ They are weakest when they are in an aqueous environment because the water disrupts the interactions. This environment has a high dielectric constant.

­ Van der Waals Interactions 

­ These are very weak interactions between any 2 atoms.

­ Half the distance between 2 identical atoms at minimum energy is the Van der Waals Radius 

­ The reason there are limited numbers of conformations of proteins are due to Phi/Psi angles of the peptide bonds. 

­ Peptide bonds are rigid and planar, and it is impossible for phi/psi angles to be 0 degrees because that would violate the rule that 2 atoms cannot occupy the same place.

­ Phi/psi angles can be seen via a Ramachandran Plot 

­ This plot illustrates the allowed conformations of peptides. 

­ Dark regions are allowed and white regions are not allowed.

Secondary Structure Details 

­ Alpha Helix 

­ Helical structure that is stabilized by intrastrand hydrogen bonds. 

­ Has 3.6 amino acid residues per helical turn. 

­ Side chains point toward the outside

­ Glycines and Prolines don’t want to be in alpha helices due to Glycine’s flexibility with no R­group and Proline’s bulky cyclic structure that would kink up the helix. ­ Two charged amino acid side chains three residues apart contribute to stabilization of the helix due to electrostatic interactions (residues 1 and 4). ­ Helix capping neutralizes the net dipoles of the N­terminus (positive) and C­terminus (negative) ends, which is stabilizing to the helix.

­ Beta Sheet 

­ Made up of Beta­Strands which are connected through interstrand hydrogen bonds  Residues 1 and 3 can potentially interact. 

­ Antiparallel Beta­Sheets:

   ­   More stable type: each strand is facing the opposite direction.

   ­   Connected by Beta­Turns: ​4 residues (Glycine and Proline are in these)

­ Parallel Beta­Sheets: 

   ­   Less stable of the two ­ can’t have tight turns

­ Disulfide Bonds​ (Covalent/strong) ­ can stabilize secondary and tertiary structures Tertiary Structure 

­ Combine the secondary elements previously described with the 4 weak non­covalent interactions and that makes up tertiary structure.

­ Structural Motif 

­ Folding pattern involving 2 or more elements of secondary structure

­ Beta­Alpha­Beta Loop, Alpha­Alpha Corner, Beta­Barrel, etc.

­   An independent folding unit on a single protein is called a Domain 

­ These are connected via covalent/strong bonds, thus, they cannot be separated by Reducing SDS­PAGE, but subunits CAN be separated.

­ Advantages: A single protein with multiple domains is more efficient ­ can have multiple different functions in one, and it has more points of regulation.

­ Disadvantages: If one of these proteins denatures, multiple functions are lost, and if it becomes mutated, it could have even more destructive effects.

Protein Quaternary Structure; Protein Folding 

       ­     ​Quaternary Structure 

­ Applies to a protein when 2 or more polypeptide chains (identical or different) come together.

      ­    Multimers​ are multisubunit proteins 

­ These can sometimes be better than single polypeptide proteins because as one subunit performs its function, it can increase the affinity for the other subunits and therefore has more cooperativity/regulation.

­ Multi­domain multimers have lots of diversity.

­ However, sometimes it is better to have multiple copies of a small protein so there is less chance for mutation and less risk for error due to less protein to fold (Tobacco mosaic virus)

­ Quaternary Structure is determined by the same 4 non­covalent interactions as secondary and tertiary structure

­ If misfolded or incompletely folded proteins hang around for too long, they aggregate and cause damage (disease) 

­ Autophagy or ubiquitin­proteasome systems take care of these incorrectly folded proteins as a last resort.

­ Proteins are constantly moving...they are dynamic 

­ Bonds are always vibrating

­ Denaturation​ is the unfolding of a protein’s tertiary structure 

­ Protein usually loses its function

­ Can be reversible, but mostly irreversible

­ When a protein is heated, energy is increased and the bonds start moving more, causing the protein structure to loosen and unfold. 

­ Hydrophobic interactions are more strong and static at low temps, but when temp increases, they move more and become weaker, loosening structure.

­ Levinthai’s Paradox 

­ Folding is non­random or else it’d take forever to fold proteins due to the immense amount of conformations to sort through.

­ Therefore, proteins follow a distinct, non­random path.

­ Protein folding can be represented by a free energy funnel 

­ Because there are so many different conformations for a polypeptide to choose at the beginning, the entropy (randomness) is high and the free energy is also high. BUT, the entropy of water is low!

­ This fact drives the folding of the protein in order to increase the entropy/disorder of the system by releasing water molecules when the protein becomes more native. This decreases the entropy and free energy of the protein though.

­ Urea and Mercaptoethanol (BME) are used to denature and reduce proteins. ­ Urea disrupts hydrogen bonds and denatures proteins while BME converts disulfide bonds to thiols.

­ Ways to see the foldedness/unfoldedness of proteins: 

­ Intrinsic Tryptophan Fluorescence​ → Fluorescence shifts if denaturation occurs and Trp is no longer buried in the core.

­ Circular Dichroism​ → See how the backbone will bend polarized light. If denatured, helices and sheets will bend it much differently.

­ A sigmoidal curve graph can be made to view the thermal or chemical denaturation of a protein vs temperature or concentration of denaturant.

­ The Tm or Cm is the temperature or concentration of chemical denaturant that causes half of the protein to be denatured/unfolded

­ If you lower or raise the pH, a protein will denature quicker when exposed to heat or denaturant. 

­ R­groups become protonated or deprotonated and charges change, causing weak interactions to already be weakened (for example salt bridges).

­ Chaperones​ are proteins that help partially or improperly folded proteins fold correctly. ­ Some proteins aggregate before they even fold due to the extremely fast process of protein synthesis.

­ Chaperones can either stall the time (Passive) or stall the time AND use energy to assist in folding (Active) 

­ Hsp70 (Passive)​ has a hydrophobic region that binds to exposed hydrophobic regions of denatured or partially unfolded proteins to “hide” the region from aggregation. It “buys time” for the protein to keep it from folding incorrectly.

­ Chaperonins (Active)​ “hide” the unfolded protein AND use ATP Hydrolysis to facilitate proper folding and the release of the now folded protein

­ If too many beta­strands from multiple polypeptide chains sit around in a partially unfolded state, they interact with each other and create a long line of beta­sheets called Amyloid Fibrils​, which are very bad and linked to diseases → the aggregates can lead to prion disease. 

Chapter 5 ­ Protein Function (Week 4)

Oxygen Binding Proteins 

­ Ligands are molecules transiently bound by proteins for those proteins to perform their functions. 

­ Ligands bind at the binding sites of proteins

­ When a ligand binds, there is usually a conformational change that takes place in the protein in order to allow tighter binding of the ligand...this is called an induced fit. 

­ If the protein is a multimeric protein, the conformational change resulting from this binding can affect the conformation of the other subunits. 

­ Oxygen is basically insoluble in aqueous solutions and can’t easily reach tissues on its own.

­ Transition metals such as iron (Fe2+) have high affinities for binding oxygen. ­ These ions if free, however, can cause free radical reactive oxygen species that can have detrimental effects, SO...they need to be “protected.” 

­ Heme​ ​is a prosthetic group which contains iron 

­ Heme is made up of what is called a Porphyrin Ring​ System which contains the iron atom in the middle. This has 6 bonds to it: 4 of which are nitrogen atoms from the flat porhphyrin ring, and then 2 that are “open” for a proximal histidine and oxygen to bind.

­ The Fe3+ state of iron does not bind oxygen, so the nitrogen atoms around the iron atom keep it in the Fe2+ state.

­ As a result of “sequestering” the iron atoms by having hemes in the middle of proteins, the free radical reactions are mostly prevented. 

­ A distal histidine in the structure acts as a steric hinderer to CO, helping keep it from taking the place of oxygen.

­ Protein­Ligand­Interactions​ ​can be described quantitatively: 

­ The fraction of ligand binding sites bound can be shown by a hyperbolic curve. ­ [L] represents concentration of ligand

­ Theta (θ)/Fraction Bound = # of proteins bound to ligands over # of all ligand­binding sites 

­ A more practical equation is: θ = [L] / [L] + Kd 

­ Kd is the concentration of ligand at which half of the binding sites are bound. ­ The lower the Kd, the more binding affinity a protein has for that ligand, and vice versa. This is because it takes less concentration of ligand to bind 50% of the binding sites.

­ There can NEVER be 100% binding sites bound!!!

­ The distal histidine​ on the heme structure impairs CO binding through lowering the affinity of the binding site to it. 

­ Due to steric hindrance by this histidine, the CO won’t bind straight on like it normally wants, instead it is forced to an angle, which makes the Kd a lot higher, thus lowering the affinity for CO to bind.

­ Oxygen prefers to bind at an angle and is therefore not affected by this.

­ Due to proteins being dynamic and always in motion, either cavities develop, or the distal histidine rotates out of the way, allowing oxygen to get into the heme structure and bind. 

­ A very important protein for transporting oxygen in your blood is Hemoglobin

­ Oxygen is basically insoluble in aqueous solutions and can’t travel through blood on its own ­ that’s where Hemoglobin comes in to save the day!

­ Hemoglobin 

­ This is part of the globin family of proteins which all primarily transport oxygen. ­ Hemoglobin is a heterotetramer, containing 4 subunits, each containing a heme group to carry oxygen.

­ Myoglobin is another oxygen transporter, however it only has one site of binding (high affinity) for oxygen.

­ When compared to myoglobin, each subunit of Hemoglobin has a closely resembled heme structure. It is structurally conserved.

­ The binding of oxygen stimulates conformational change in the protein structure. 

­ The T­state​ (tense state) is when there is a high Kd for oxygen → lower affinity ­ The T­state is stabilized by ionic interactions and other non­covalent interactions, and the middle cavity (between Beta­subunits) is more open.

­ The R­state​ (relaxed state) is when there is a low Kd for oxygen → higher affinity ­ When the R­state forms, ionic interactions are broken, and the cavity in the middle tightens, increasing affinity for oxygen.

­ The huge conformational change that occurs in the protein is due to the subtle side chain movements at the oxygen binding site from the oxygen binding to it. ­ Upon oxygenation, the iron atom becomes in line with the plane of the Heme, which causes the proximal histidine to move closer to the Heme, thus shifting position of amino acid residues near it. When those shift, the interface structures between the subunits of hemoglobin are altered and the protein conformation changes.

­ Hemoglobin is a cooperative oxygen binding protein 

­ When oxygen binds to one of the subunits, it impacts the ability of another to bind oxygen. Therefore, it regulates the affinities of the other subunits and is considered an allosteric protein.

­ The first binding will usually be the hardest, but then the subsequent binding should progressively get easier. 

­ Hemoglobin is perfectly suited to transport oxygen from the lungs to body tissues ­ Myoglobin has only one subunit and therefore binding of ligand cannot affect binding at another subunit, therefore the binding curve for oxygen would NOT be sigmoidal. 

­ We WANT a sigmoidal curve so that there is cooperativity taking place to change the conformation of the protein in order to have high affinity for oxygen in the

lungs (98%) and then lower affinity for oxygen in the tissues (60%) so oxygen can be “offloaded” (38%). Hemoglobin gives us this curve. → The curve is represented in the lecture slides from class.

­ We never know what combination of states (T or R) hemoglobin is in, but it is most likely like a “faucet” rather than a “light switch.” ­ This means hemoglobin is most likely constantly sampling between the T and R states due to proteins being dynamic.

­ The Concerted Model (MWC model) states that there is a smaller set of possibilities of states in the protein that can interconvert.

­ The Sequential Model basically says that there is every single possibility of combinations and there are lots of interconversions dictated by environment (most accepted theory).

­ Hill Plot 

­ This describes cooperative binding quantitatively in a graph 

      ­     θ = [L]n / [L]n + Kd where “n” is the number of binding sites

­ A positive slope = positive cooperativity, negative slope = negative cooperativity, and slope of 1 = no cooperativity.

­ Log (θ / 1­ θ) = n log [L] ­ log Kd → rearranged ­ Hill Equation 

­ pH can change the affinity for oxygen in hemoglobin 

­ If it is lowered, there will be less affinity for oxygen ­ His 146 has (+) charge. ­ If it is raised, there will be more affinity for oxygen ­ His 146 has neutral charge. ­ Carbonic Anhydrase generates HCO3­ and protons ­ forms positive charge.

­ CO2 reacts with the N­terminal amino group of the hemoglobin subunits and stabilizes T­state. 

­ The Bohr Effect​ is the effect of [H+] and [CO2] binding on oxygen affinity.     ­     H+ and CO2 binding decrease affinity (tissues)

    ­     CO2 excreted and pH rises ­ increases affinity for oxygen. (lungs) ­ BPG ­ 2,3­Bisphosphoglycerate 

­ BPG regulates O2 binding by binding to the central cavity of hemoglobin, stabilizing the T­state, making it harder for O2to bind.

­ This is produced in HIGHER amounts at higher altitudes….uh, why..? ­ ***See the graph in the last set of lecture notes on page 9***

­ At a high altitude with 5 millimolar BPG, you only offload 30% O2to the tissues, so BPG kicks up to 8 millimolar...

­ This shifts the binding curve for oxygen to the right, which causes affinity for oxygen to decrease a little in lungs, and the offloading is set back at a basically ideal 37% offload in the tissues.

­ When oxygen binds to hemoglobin, BPG binding site at the core disappears. ­ When oxygen is released from hemoglobin, BPG binding site at the core appears.