BMB 251 1st Edition Lecture 11 Outline of Last Lecture I. PhotobleachingII. FRABIII. Electron Microscopesa. Immunogoldb. SEMc. TEMIV. Cryo-Electron Microscopya. Single particle reconstructionOutline of Current Lecture V. Proteinsa. Binding sites b. Ligandc. AntibodiesVI. Equilibrium constant (K)VII. Enzyme CapabilitiesVIII. LysozymeIX. CoenzymesX. Allosteric bindinga. Negative regulators/inhibitorsb. Positive regulators/cofactorsCurrent Lecture- All protein molecules bind to other molecules; protein’s physical interaction with other molecules determines its biological propertieso Hydrophobic effect is the main driving force in protein folding  folding completely determines the 3D conformation of a protein, creating a unique binding sites o Binding always shows great specificity: each protein molecule can usually just bind to one or a few molecules out of he thousands it encounterso Not only the shape contributes to the specificity in binding, but also the protein’s physical and chemical interactions which make it stand out from others o **Ligand: substance bound to protein (ex. Ion, small molecule, another protein, etc.) Successful binding depends on weak, noncovalent bonds- Binding site: region of protein that associates with ligand; usually consists of a cavity in the protein surface formed by a particular arrangement of amino acidsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Proteins have impressive chemical capabilities because the neighboring chemical groups on theirsurface often interact in ways that increase chemical reactivity of side chainso Interaction of two neighboring of polypeptide chain may restrict access of water molecules to binding sites, which compete with ligands by forming H-bonds to the protein surface (energetically unfavorable for a single water molecule to break away from the rest to seep in)o Clustering of neighboring polar amino acid side chains can alter the reactivity  Ex. If protein folding forces together many negatively charged side chains, affinity of the site for positive ion greatly increases- **Surface of each protein = unique chemical reactivity that not only depends on which amino acid side chains are exposed, but also on the exact orientation relative to one another- SH2 domain allows protein-protein interactions; 3 main ways of binding:o Surface-string: SH2 domain recognizes phosphorylated polypeptide loop of second proteino Helix-helix (coiled coil): two alpha helices (one from each protein) pair togethero Surface-surface: precise match of two rigid surfaces; bonds are very tight (many bonds within surfaces) and are highly specific (usually just one partner)- Antibodies: proteins produced by immune system in response to foreign molecules, such as those of the surface of invading microorganismo Tight, selective binding to particular target molecule  inactivating target molecule directly or marking it for destructiono Target = antigen; antibody recognizes it with remarkable specificityo Y-shaped molecules with two identical binding sites complementary to a small portion ofthe surface of an antigen molecule- Equilibrium constant (K) can be determined from concentrations of ligand, antibody and antibody-ligand complexes o Determines the strength of the bindingo K for A + B  AB in L/moles will have half the binding sites occupied by ligands when ligand’s concentration = 1/Ko Equilibrium constant increases as binding strength increaseso Direct measure of free energy distance between bound and free states - Enzymes speed up reactions without being changed themselves (aka catalysts)o They lower the activation energy of the system via the specific location they are placed within the amino acid sequenceo They cannot affect the system’s free energyo They are able to speed up the rate of reaction by HUGE magnitudes (in some cases making a reaction process go from taking years to mere milliseconds)- Turnover number: maximum rate of how rapidly the enzyme can process the substrate molecule divided by enzyme concentration; often about 1000 substrate molecules processed per second- Km: concentration of substrate allowing reaction to proceed at half its max rate (0.5 Vmax)o Low Km value means enzyme has reached its max catalytic rate at low concentrations of substrate and enzyme binds to substrate very tightlyo High Km = weak binding- Activation energy: free energy required to attain the most unstable transition state; major determinant of reaction rateo Enzymes have high affinity for transition state of substrate rather than stable form. So they greatly decrease the activation energy of reaction- Lysozyme: catalyzes the cutting of polysaccharide chains in cells walls of bacteriao Causes cell wall to rupture and cell to bursto Catalyzes hydrolysis  adds water to single bond between two sugar groups in polysaccharide chain, causing bond to breako Binding of lysozyme to substrate = noncovalent bonds- Three steps in the enzymatically catalyzed reaction:o Enzyme stresses bound substrate  shape of one sugar resembles the transition state formed during the reaction via noncovalent bodso Negatively charged aspartic acid reacts with C1 carbon atom on distorted sugar and glutamic acid donates proton to oxygen that links sugar to its neighbor  breaks sugar-sugar bondo Aided by negatively charged glutamic acid, water reacts with C1 carbon atom to displace aspartic acid side chain and competes hydrolysis; acids are returned to their original states and products diffuse out separately- Proteins often use small nonprotein molecules to perform functions that would be difficult/impossible for amino acids to do by themselves o Ex. Retinal embedded in protein rhodopsin  helps detect light in retina o Ex. Hemes in protein hemoglobin  gives it its red color and picks up oxygen in lungs to release it throughout tissues- Conenzymes: organic molecules in enzymes that assist in hydrolysis reactions o Enzymes are bigger molecules, while coenzymes tend to be smaller molecules, but enzymes do not always contain all the necessary amino acids needed to undergo a reaction-sometimes they cannot be synthesized by the body and must be taken in through dieto Ex. Biotin (transfers carboxylate from one molecule to another); is a vitamin Must get biotin from our diet/cannot be synthesized in humans- Molecular tunnels: evolved in enzymes