Protein Ligand Interaction Protein Function Hemoglobin Biochem 4511 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Ed Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Protein Ligand Interactions A ligand is any molecule that binds to a protein Typically ligand binding is reversible transient and specific When the protein is an enzyme often the ligand is a substrate Apoprotein p p is a p protein with an empty p y binding g site Holoprotein or holoenzyme is the ligand bound form of protein Protein Ligand Interactions Interactions between protein and ligand are comprised of the same elements commonly found in a folded protein Hydrogen bonds Hydrophobic interactions Electrostatic interactions Dipole dipole interactions Rarely covalent interactions find fi d an example l iin thi this llecture t Specificity Lock Key vs Induced Fit Lock and Key Hypothesis Induced Fit Hypothesis Binding site of protein is a perfect match for the substrate Binding site of protein is similar to the substrate and when bound subtle changes occur in the structure of both species Ligand Specificity Proteins specifically recognize particular ligands Lock and Key Hypothesis Specificity of a protein for its ligand arises i ffrom complementary l t shapes h Geometric complementarity Si and Size d shape h off cleft l ft and d liligand d Electronic complementarity p y Dihidrofolate reductase with substrates NADP red and tetrahydrofolate yellow yellow Recognizes hydrophobicity charge hydrogen bonding Induced Fit Hexokinase Hexokinase No substrate bound open cleft Glucose bound closed cleft Binding of glucose and ATP changes conformation of hexokinase Quantitative Description of P t i Li Protein Ligand d Interactions I t ti k1 P L PL k 1 Ka k1 k 1 PL P L Association constant Kd k 1 k1 P L PL Dissociation constant Dissociation Constant Kd k 1 Kd k1 P L PL fraction bound PL bound P PL total Since PL P L Kd L Kd L When L Kd 50 of binding g sites are occupied p half saturation If L Kd more is bound If L Kd less is bound Dissociation Constant Kd Higher Kd is indicative of weaker interactions with ligand L Myoglobin a muscle oxygen carrier Prosthetic group heme Heme prosthetic group H Heme is i a porphyrin h i that h chelates h l iron i Residues two His are key to regulating heme and oxygen Question Which parts of the heme do you predict to be exposed to solution Myoglobin binding Oxygen Mb O2 MbO2 Kd P L PL Mb O2 Kd MbO2 Recall that Y L Kd L O2 pO2 for a gas Can rearrange to Y pO2 Kd pO2 So fraction bound is a function of affinity and O2 concentration For myoglobin Kd 2 8 torr pO2 in capillaries is 20 40 torr Hemoglobin heterotetramer Primary O2 carrier in blood in red blood cells Hemoglobin 2 subunits grey light blue 2 subunits red dark blue 4 different O2 binding sites Deoxyhemoglobin no O2 Oxyhemoglobin bound O2 Myoglobin and hemoglobin subunits have similar structure and function Yet sequence identity is only 18 Sequence homology and structure function Invariant identical in all Hbs and Mbs Identical in the compared Identical in Hb Alignment and comparison of sequences from different proteins or species can imply functional residues Molecular Evolution 1 1 billion years ago Comparison of primary sequences can imply evolutionary relationships l ti hi and d predict di t properties ti with ith caution ti Known rates of mutations can imply age of branching F Fraction of occu upied sittes Oxygen affinity Myoglobin Hemoglobin Mb Kd 2 8 torr Hb Kd 26 torr Why hemoglobin i so is inefficient as compared to myoglobin Note the hemoglobin curve shape is sigmoidal sigmoidal not hyperbolic High affinity at pO2 of lungs 100 torr low affinity at pO2 of tissues 20 40 torr Hemoglobin allostery Hemoglobin is a heterotetramer consisting of 2 and 2 subunits H Hemes are separated t d too t far f to t directly di tl affect ff t each h others th affinity to oxygen How Allosteric long distance effects Allosteric regulation Allostery regulation of a protein by ligand binding at a distant site other than the protein s active site Such site is called allosteric Allostery comes from the Greek allos other other and stereos object protein s activity are allosteric Ligands that enhance the protein s activators Ligands g that decrease the p protein s activity y are called allosteric inhibitors Long range g g allosteryy is especially p y important p in cell signaling and metabolism Allostery long range conformational change explains cooperative binding a green pink regions with low affinity Spend p most of the time in conformation incompatible with binding b Binding g of first ligand g induces conformational change in 2nd unit overall more stable structure with higher probability of binding in pink c 2nd ligand binds with higher affinity Remember allostery can increase or decrease affinity Each Subunit contains a Heme F Four hemes h means four f potential t ti l oxygen binding bi di sites and numerous interactions between subunits Structural Changes in Hb T and R states R T Purple P rple R high affinit affinity state Blue T low affinity state Below alternative view Seemingly subtle conformational changes at tthe eb binding d g ssite te a are e ttranslated a s ated to overall dramatic structural changes in the whole protein Binding of Oxygen Changes Hemoglobin Structure 1 Binding of oxygen and its bonding to His64 repositions iron in the heme ring now in in plane plane 2 As a result an adjacent helix is also repositioned Structural changes at the interface These subtle changes propagate through altered interface interactions leading g to g global rearrangements in Hb quaternary structure The Two States of Hemoglobin Binds oxygen poorly Binds oxygen readily Flat heme and shifted helix The Two States of Hemoglobin Binds oxygen poorly Binds oxygen readily Flat heme and shifted helix F Fraction of oxyg gen occu upied siites Hemoglobin Oxygen affinity R state Myoglobin T state 1 Carbon Monoxide CO bi d tto Hb 250 times binds ti more efficiently than O2 fewer sites available for O2 binding 2 Additionally binding of CO iinduces d ttransition iti to the high affinity R state CO binding is nearly irreversible and strongly delays release of O2 Fraction of o oxygen oc ccupied s sites Carbon Monoxide CO p and Interferes with O2 release Competes Breathing g 100 oxygen yg reduces half life of COHb from 7 hours to 10 20 minutes CO poisoning COHb