BIO515 Exam 1 Review - Biomolecules PDF

BIO515 EXAM 1 REVIEW - Major Concepts (Lehninger 7th Edition) Chapter 1: Biomolecules Biological macromolecules: long chains containing many freely rotating single bonds, allowing tremendous conformational flexibility. Macromolecules can form infinite conformations yet actually fold into and hold...

BIO515 EXAM 1 REVIEW - Major Concepts (Lehninger 7th Edition) Chapter 1: Biomolecules Biological macromolecules: long chains containing many freely rotating single bonds, allowing tremendous conformational flexibility. Macromolecules can form infinite conformations yet actually fold into and hold specific conformations. - The same four non-covalent interactions allow macromolecules to reversibly bind specific ligands. - Structures of macromolecules determine their function. Ligands bind into the cleft by shape and charge complementary using weak interactions; hydrogen bonding, ionic interactions etc. its reversible because the interactions are so weak. Chemical bonds involving Carbon, Oxygen, Hydrogen, and nitrogen form the basis of cellular life. The shape of small and large molecules is determined by the type and configurations of the covalent bonds that they contain. Noncovalent weak interactions become increasingly important for the shape of the larger molecules. Lysozymes: a protein in human tears and egg whites hills bacteria by cleft precisely matches shape of the molecule that make bacterial cell walls, specific atoms in the right place within cleft break the covalent bonds of the bacterial cell wall Covalent bonds: Sharing of electrons, the strongest interaction, valence electron, 2 electrons one from each atom. The bond is polar or non-polar based on the atoms involved. - Nonpolar à C-H, C-C - Polar à O-H, N-H, C-O, C-N – partial charges - Backbone of all major macromolecules - Single bonds rotate double bonds do not. Ionic bonds: attraction of opposite charges (+,-) - Salt bridges - Attraction is stronger when they are closer to each other. Hydrogen bonds: sharing of H atom, hydrogen acceptor and donor Donor: O-H and N-H, the O or the N are highly electronegative which leading to formation of partial positive charge Acceptor: The oxygens in polar covalent bonds such as C-O and C=O and nitorgens Hydrophobic interaction: Interaction of nonpolar substances in the presence of polar substances espcially water) Hydrophobic effect: The energetic preference of nonpolar molecular surfaces to interact with other nonpolar molecular surfaces and thereby to displace water molecules from interacting surfaces. - Lipids molecules disperse in the solution; nonpolar tail of each lipid molecules is surrounded by ordeed water molecules - Enropy of the system decreases - Unfavorable state  - Micelles are formed (ball of all hydrophobic groups are sequestered from water; ordered shell of H2O molecules is minimized and entropy is further increased. - Hydrophobic effect favors ligand binding because he enzyme substrate interaction stabilized by hydrogen bonding, ionic , and hydrophobic interactions - Binding sites in enzymes and recptors are often hydrophobic, such sites can bind hydrophobic substsrates and ligands such as steroid hormones - Contribute to protein folding and stability Van der Waals Interactions: Interactions of electrons of nonpolar substances (stacking) Thermodynamics DG= DH-TDS G is the Gibbs free energy: the enrgy in asystem that is avalible to do work H is enthalpy: it descirbes the numbers and kinds of bonds T is the temperature S is the entropy it describes the randomness or disorder in a system If DG is negative the reaction is favored (spontaneous) If DG is positive the reaction is not favored (not spontaneous) DG is usually negative if entropy increases Exergonic reactions RELEASE energy, products have LESS energy than reactants (down the hill) ex. Hydrolysis if ATP Endergonic reactions require the INPUT of energy products have MORE energy then reactants (up the hill) ex. Synthesis of sugars from CO2, H2O using sunlight, during photosynthesis OIL RIG à Oxidation is losing electrons; Reduction is gaining electrons. Combustion is – DG release energy it is favorable. Cellular respiration is favorable increase in molecules -DG. Chaper 2: Water Water dissolves substance by forming a sphere of hydration = many H-bonds around substance in a 3D sphere, crystal lattice is reduced Entropy of water molecules: Water in bulk has a lot of enropy each water molecule can rotate around in any direction and make h-bonds with other molecules. Water and lipids: Water molecules near the lipid have less degrees of freedoms- since they cannot turn around to form h-bonds with the lipids they move less and tend to form a static layer around the lipid, much less entropy everything becomes veryyyy ordered. Hydrogen bonding in water and properties that result: Hydrogen bonds in water give it all its properties of cohesiveness, adhesiveness, high heat of vaporization. Waters highly mobile in liquid, tumble past each other, forming, breaking and reforming bonds as they go. H-Bonds are individually weak but collectively strong Hydrogen bonds with polar solutes (N-H, C-N, O-H, C-O bonds) * Water is a good solvent for charged and polar substances-hydrophilic à amino acids and peptides, carbohydrates. and it is a poor solvent for nonpolar substance hydrophobic. Hydrophobic à aromatic moieties, aliphatic chains, nonpolar gases Interactions with charged solutes Osmosis is the movement of “free water: across a semi-permeable membrane. - Water moves towards the side with more solute. - Cells have adaptations to deal with osmotic pressure (water rushing into a “saltier cytoplasm that solution around it” - Osmolarity depend on the number of solute molecules (moles) and not on their mass. Water can ionizeà Products are proton (H+) and a hydroxide ion (OH-) ionization of water is a rapid reversible process. - Equilibrium is strongly to the left. [" ! ][$" " ] 𝐾𝑒𝑞 = ["%$] 1.8 ∗ 10&'( 𝑀 𝑎𝑡 25°𝐶 Ionic product of water = 1*10^-14 M pH is defined as the negative log of H+ concetration pH= -log[H+] = log(1/[H+]) - Hence a change of 1 pH unit means a 10 fold change in concentration of H+, 2 pH is 100 fold change - Biological molecules are very sensitive to pH because both their structure and activity are dramatically affected by even small changes in pH Acids à Produce H+ in aqueous solutions, release H, proton donor (H+), electron pair acceptors, H+ is a strong acid Bases à Produce OH- in aqueous solutions, release OH, proton accacceptors, electron pair donors, NaOH is so strong base - Weak acids and bases don’t completely ionize, The dissociation is determined by the acid dissociation constant Ka pKa=-log Ka low pka = stronger acid a high Ka means the equilibrium is shifted to the right = stronger acid [)" ] pH=pka+log[")] A-= proton acceptor, HA= proton donor Dissociation constants (Ka) and pKa (*) At pHs below pKa, HA>>A-  At pHs above pKa, A->>HA Buffering (*) - Buffers resisr change in pH - At pH=pKA there is a 50;50 mixture of acid and anions forms of the compound - Buffering capacity is lower when the pH differs from pKa by more than 1 pH unit - When you add H+ or OH is a buffered solution near pKA the solution captures the H+ in its own equilibrium - This goes on until you’ve added so much H+ that all of the weak acid or its conjugate base are exhausted and buffering power is lost. Buffer systems - Maintenance of intracellular pH is vital to all cells - In cells: buffer systems in vivo are mainly based on phosphate, bicarbonate, histone. - In lab; Buffer systems in vitro are often based on sulfonic acids of cyclic amines or primary amines. Chapter 3: Amino Acids, Peptides, and Proteins (important chapter) Amino acids à All amino acids contain an amino group and a carboxylic group (-COOH)is acidic ( it releases a proton to become -COO) Characteristics unique to each amino acid which gives the aa its properties - Proline is the only AA where R group is connected to backbone - All aa are chrial (except glycine) Nonpolar groups – like to make hydrophobic interactions with each other or wih ligands, tend to cluster within the interior of proteins (away from water), G and P are alpha helixes breakers where a-helix end or turn around, M is the starting amino-acid of every protein that is initially translated. Aromatic R Groups – non polar, hydrophobic interactions, F is more hydrophobic then Y and W, often form stacking interactions with each other or with NA, Y is a site of phosphorylation at the -OH. Polar uncharged R groups à These amino acids side chains can form hydrogen bonds, Cysteine can form disulfide bonds. Negatively charged groups à weak acids side chain pKa ~4 Positively charged groups à ionizable side chains, basic, H pKa near pH 7 means at physiological; pH histidine side chain might be partially pronated. Hydropathy à how likely that amino acid will be happy to interact with water (surface of the protein), a negative -G indicates favorable interaction (exterior), -deltaG unfavorable interaction (hydrophobic) (interior) - Proline, glycine, tyrosine, tryptophan, Serine, threonine, asparagine glutamine, lysine, histidine, aspartate, glutamate found on surface. Isoelectric Point, pI is the pH at which the molecule is electrically neutral (no net charge) For amino acids without ionizable side chains, the Isoelectric Point (equivalence point, pI) is eh average of the pKas of the carboxy and amino groups pI = pK1+pK2/2 Deprotanted = -1 Protanted = +1 When the pH matches the AA pI the aa is least soluble in water, aa does not migrate in electric field, the same is true for peptides and proteins. Peptides are small condensation products of amino acids, amino acids are referred to as residues in peptide or protein, the start is N-terminus, the end is C-terminus. To study a protein one must purify it Step of protein purification: 1. Breaking cells/homogenization/extractionà Blender, osmotic shock, sonicator, glass beads, enzymatic treatment 2. Differential centrifugation à Separates cell components on the basis of size and density, the larger sediment forms a pellet at the bottom of the tube while the smaller components remain in suspension above, called the supernatant. 3. Salting-out/dialysis and concentration à Hydrophobic aggregation either by unfolded proteins or by removing the water that surround and solvates the protein which may make protein stick together cause there is no water. a. Solvation: certain ions may be required for the protein to be souble (“salting in”) b. Aggregation: certain salts disrupt water solvent shell and cause proteins to precipitate (‘salting out”) c. Hydrophobic aggregation is enhanced when the pH of the solution is close to the proteins pI, that is because proteins tend to be less soluble at pI, because at pI a protein carries a net charge of 0, so the protein molecules don’t repel eachother, hence they aggregate with each and precipiate 4. Ion-exchange, à at a pH BELOW the pI the protein gains protons and is positively charged, while at a pH ABOVE the pI the protein loses protons and is negatively (-) charged a. Two types of ion exchange: i. Anion (-) à Solid phase is (+) charged binds negatively charged proteins. ii. Cation (+) à Solid phase is (-) charged so it binds positively charged proteins. b. How to elute proteins? à either change the salt concentration to elute bound proteins, ions in salt bind to the protein and neutralize its charge, so that the protein is released from the gel. OR can change the pH to elute bound proteins because pH can affect the charge on the protein (depends on a proteins pI) 5. Affinity chromatography à Molecules that can intercat with the covalently bound ligand. The stringer the interaction the longer it takes the protein to go through the column, if something has too high an affinity for a ligand then it will not dissociate and the purification is not effective. 6. Column chromatography à Solid phase remains in place and the mobile phase carries the mix through the solid phase. 7. Gel-filtration chromatography (size exclusion) à The smaller the molecule he longer it takes because the small molecules get stuck inside tge beads and the larger molecules travel past the beads 8. SDS-PAGE(also isoelectric focusing and 2D PAGE) à Protein samples placed at the top of gelk with pores about the size of protein molecules, small molecules move through gel more easily, so the protein gets sperated by size a. SDS: bind proteins and gives all the proteins a large negative charge proportional to the MW ~charge/mass ratio, and it also denatures them, thus easier to interpret migration on a gel  b. What information do we get from SDS-PAGE? à Purity of protein smaple and determine/confirm MW of protein of intrest. 9. Functional assay à final steps of purification, ex. Lysozyome activity assay à put purified protein sample ona a paper disk, place disk on a petri dish of bacteria, lysozyme activity: size of the “lysis” halo around the disc, representing area of inhibition of bacterial cell death. 10. Mass spec à modern methods to determine the accurate MW of a protein (MALDI- MS) and to sequence a short peptide or identify a protein (MS/MS) these can be doen using minute amounts of material and often is good enough to identify an unknown protein EXAMPLE OF A WORKFLOW à Affinity chromatography for ligand binding, then ion exchange chromatography to separate by charge, size exclusion to separate by size. Amino acid homology symbols *= Invariant conserved aa : - conservative substitutions (same aa family) No symbol: nonconservative substitutions (diff aa family) Chapter 4: The Three-Dimensional Structure of Proteins Primary structure à linear sequence of amino-acids in a peptide/protein ex. -Ala-Glu-Val- Asp Secondary structure à a-helix and B sheet Tertiary structure à The combination of a-helix and b-sheet Role of weak interactions in protein structure: Overall, weak interactions within protein structures collectively contribute to the stability, conformational flexibility, and function of proteins. They enable proteins to fold into their native structures, interact with other molecules, and carry out specific biological functions essential for cellular processes. Peptide bond is rigid and planar -à peptide bond removes + and – charges from the functional groups, the carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, setting up a small electric dipole. Virtually all peptide bonds in proteins occur in this trans configuration, peptide bond is partially double bonded = rigid, rotation occurs in the bonds around the peptide bonds (single bonds). Phi angle: around C-N-Ca-C Psi angle: around N-Ca-C-N Secondary structure: refers to a local spatial arrangement of the polypeptide backbone. Structure forms form hydrogen bonding of the backbone not thru the side chains, the C=O and the N-H of the peptide bonds can form H-Bonds Every backbone N-H group is an H bond donor and every backbone C=O group is an H bond acceptor. (except prolines N) a-helix: helical shape stabilized by hydrogen bonds between backbone atoms of nearby residues. C=O and N-H groups, backbone coils into right-hand helix (usually). H-bond between C=O of aa #1 and N-H of aa #5. Properties: 1. Different amino-acids have different prosperities to form a-helices 2. R-group interactions can stabilize or destabilize an a-helix, especially 3-4 AAs apart 3. An a-helix has a dipole-which favors certain amino-acids near the ends 4. Amphipathic helices possible (different “faces” of a helix) à both polar or nonpolar, one face of the helix may havr polar side-chains while the other “face has nonolar side chains 5. Alanine is most likely aa to be in alpha helices because the R group is small 6. Proline destabilizes a-helices because the backbone is constrained such that it cannot adopt the right dihedral angles for an a-helix, the N has no H to contribute to H-bond. 7. Glycine is to flexible because it doesn’t have a large side chain. 8. Negatively charged aas are more likely near the N-term because it is positively charged and positively charged aas are more likely near the C-term. b sheets: sheet-like shape composed of strentches of AAs that pair up in parallel or parallel arrangement. Stabilized by hydrogen bonds between backbone atoms of adjacent segments that may not be nearby (in sequence) 1. Two or more extended chains running alongside each other 2. Held together by H-bonds between backbone C=O and N-H of adjacent strands 3. “pleated” due to planar geometry around the partially double peptide bond 4. Side chains alternate above and below sheet 5. Parallel à B sheets the H-bonded strands run in the same direction resulting in bent H-bonds (weaker  6. Antiparallel à B sheets the H-bonded strands run in opposite directions resulting in linear H-bonds (stronger) The B turn: A 4 residue stretch that induces a sharp hairpin turn in the protein. Frequntly connects anti-parallel beta-sheets and/or a-helices. Usually has a proline (AA2) and glycine (AA3). First and 4th amino aid make an hbond to eachother Random coil: No particular 2° Structure- irregular arrangement of the polypeptide chain Dihedral angles and Ramachandran plots A Ramachandran plot shows he distribution of phi and psi dihedral angles that are found in a protein (or proteins) Tertiary Structure: refers to the overall spatial arrangement of all atoms in a protein- the “shape” or “fold” or “overall native conformation”. - Contains multiple elements of secondary structure: mostly a-helices or mostly B-sheets or mis of both. - Stabilized by numerous weak interactions between amino acid side chains. Can also be stabilized by disulfide bonds. - Globular: Chains folded into spherical or globular shapes-majority of proteins (myoglobin, hemoglobin) - Fibrous: Polypeptide chains arranged in long strands or sheets (keratin proteins in hair, collagen in joints) Key concept: Secondary structures like alpha helices and b sheets form based on H-bonding of the peptide backbone. IN CONTRAST with tertiary structures is based on interactions between side chains using all our types of non-covalent interactions (and disulfide covalent bonds). Quaternary structures: a protein complex made up of multiple polypeptides bound to each other, Only certain proteins form 4° structures. Hemoglobin is a tetramer. Motif: Specific arrangement of several secondary structure elements: all alpha helix, all beta-sheets, both. Can be found as reoccurring structures in numerous proteins, proteins are made of different motifs folded together. Domain: parts of a polypeptide chain that can fold independently. X-ray crystallography: purify the protein, crystallize the protein, collect diffraction, calculate electron density, and fit residues into density. Structural “resolution” is measured in Angstroms. Smallest distance you can resolve 2 separate objects. - High quality < 3 Angstroms - Low quality > 5 Angstroms NMR: Purify the protein, dissolve the protein, collect NMR data, Assign NMR signals, Calculate the structure, pros: no need to crystallize the protein and can see many hydrogens. You can get insight to see what part of the protein moves. Cryo-EM: A beam of electron is fired at a frozen protein solution. The merging scattered electrons pass through a lens to create a magnified image on the detector, from which their structure can be worked out. It reveals structures of proteins that are difficult to crystallize. Monomer: consist of a single polypeptide chain that functions as an independent unit, like insulin. Dimer: two identical or non-identical subunits. These subunits interact through various types of bonds like h-bonds, hydrophobic interactions, or disulfide bonds. Hemoglobin consisting of 2 alpha helices and 2 beta sheets. Trimer: Three subunits, collagen Tetramer: Four subunits, hemoglobin Protein unfolding: Protein denaturation (loss of all but primary structure) possible denaturants are heat, disulfide bond reduction, extremes of pH, acetone or alchol (chaotropic)à change H2O structure around the structure. - Denaturation occurs very little then all at once, unfolding is a cooperative process i.e. loss of structure in one part destabilizes another part. Protein folding: The physical process by which a linear polypeptide folds its characteristics and functional three-dimensional structure. Classical experiment by Anfinsen: What is required for protein folding? à Start by completely denaturing the protein RNase A. (RNAse A activity is easy to check). This experiment showed that the tertiary structure is determined entirely by its amino acid sequence. Removal of the denaturant refolded the protein. Levinthal’s paradox: Therefore, if a protein were to attain its correctly folded configuration by sequentially sampling all the possible conformations, it would require a time longer than the age of the universe to arrive at its correct native conformation The two main events that drive protein folding: 1. Secondary structure formation localized folding is part of the peptide. 2. Hydrophobic collapse-hydrophobic residues stick together and away from the solvent (these steps reduce the overall energy of protein until it reaches the lowest energy structure) Molecular chaperones prevent aggregation and incorrect folding by binding to and stabilizing or totally unfolded protein polypeptides until the polypeptide chain is fully synthesized. Essential they give misfolded proteins a “quiet corner” so they can fold correctly away from the mess of the cytoplasm. Mechanism of GroEL-GroES: GroEL/ES first completely unfolds the misfolded proteins, then moves the protein to a large cavity and gives it another chance at folding. Protein Misfolding: occurs when a protein fails to adopt its correct three-dimensional structure, leading to the formation of abnormal conformations. It can happen during protein synthesis and can be caused by many factors like genetic mutations, environmental stressors, or errors in the folding processes. Protein misfolding diseases: errors in protein misfolding cause the formation of disease- causing amyloid fibrils. Alzheimer’s and Parkinson’s disease are common amyloid diseases characterized by amyloid aggregation/misfolding. Prions: Proteins that when misfolded bind to and cause other copies of the same protein to misfold, if this happens and misfolding occur it can cause Kuru (humans)/mad cow disease (cows). Proteostasis: Overall term of homeostasis of protein folding, ensuring that proteins are correctly synthesized, folded, assembled, and degraded. It works as quality control within cells.