Protein Function Chapter 5 1 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 Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Protein Ligand Interactions Ligand a molecule that binds to a protein Binding Usually reversible and transient Recognition Geometric complementarity Size and shape of cleft and ligand Electronic complementarity Recognizes hydrophobicity charge hydrogen bonding Specificity Lock Key vs Induced Fit Lock and Key Induced Fit Lock Key top Induced Fit bottom Dihidrofolate reductase with substrates NDAP red and tetrahydrofolate yellow Hexokinase glucose in red Don t memorize protein identities just vocab concepts Quantitative Descriptions for Protein Ligand Interactions P L PL k1 k 1 Dissociation constant Kd k 1 k1 P L PL Meaning of Dissociation Constant Y or q fraction bound PL P PL rearranges to q Y L Kd L So Half of binding sites are occupied half saturation when Where L Kd equal amounts of bound and unbound If L Kd more is bound If L Kd less is bound L Kd Meaning of Dissociation Constant Higher means weaker interactions with ligand Lower means stronger interactions with ligand A ligand in this figure Myoglobin an oxygen binding protein Heme prosthetic group Heme is a porphyrin that chelates iron Residues two His are key to regulating heme and oxygen red Question Which parts of the heme do you predict to be exposed Myoglobin binding Oxygen Mb O2 MbO2 Kd P L PL Kd Mb O2 MbO2 q or Y total bound MbO2 Mb MbO2 O2 pO2 for a gas Can rearrange to Y Kd pO2 So fraction bound is dependent on affinity and O2 concentration For myoglobin Kd 2 8 torr Protein Function Hemoglobin Deoxyhemoglobin 2 a subunits grey light blue 2 b subunits red dark blue 4 different O2 binding sites Myoglobin and hemoglobin subunits have similar structure and function Sequence homology and structure function Invariant Identical in all Identical in Hb Alignment and comparison of sequences from different proteins or species can imply functional residues Molecular Evolution Comparison of amino acid sequences can imply evolutionary relationships and predict properties With large error bars known rates of mutations can imply age of branching Protein Function Hemoglobin Deoxyhemoglobin 2 a subunits grey light blue 2 b subunits red dark blue 4 different O2 binding sites Myoglobin and hemoglobin subunits have similar structure and function Oxygen affinity Myoglobin Hemoglobin s e t i s i d e p u c c o f o n o i t c a r F Note the hemoglobin curve shape sigmoidal not simple High affinity at pO2 of lungs low affinity at pO2 of tissues Allostery Schematic diagram of cooperative binding a green red regions low affinity b Binding of first ligand induces conformational change in 2nd unit more blue less flexibility in red c 2nd ligand binds with higher affinity in this diagram Note allostery can increase or decrease affinity Cooperativity is key Binding oxygen T state and R state 0 O2 bound 4 O2 bound Binding oxygen Allosteric regulation No oxygen All in T state If 1 oxygen weak binding Transition with oxygen to R state additional oxygen molecules bind easily Hemoglobin Oxygen affinity R state T state s e t i s i d e p u c c o f o n o i t c a r F Structural Changes Purple R high affinity state Blue T low affinity state Below alternate view Think about nature of change absolute identity of AAs not important Structural Changes Change propagates through altered interactions Think about nature of change absolute identity of AAs not important Carbon monoxide CO Trapped in R state Allosteric regulation CO locks structure into R state blocks release of O2 Half life of COHb 2 7 hours at atmospheric O2 Treatment with 100 oxygen pressurized reduces half life to 10 20 minutes Carbon Dioxide CO2 Increase O2 release directly Carbamates form salt bridges that stabilize low affinity T state High CO2 Transition to T release x allosteric regulation CO2 is usually high in tissues where O2 is needed Bohr Effect Christian Bohr 1904 pH and respiration HbH O2 HbO2 H CO2 H2O HCO3 H x binding is also regulated by pH Low pH more H more HbH bound less O2 bound Indirect CO2 mechanism through blood buffering More CO2 more H less O2 bound different from prior slide BPG Dynamic regulator of oxygen BPG Allosteric regulator of hemoglobin BPG T state low affinity Blue patch of basic residue s R state no binding site Sickle Cell Anemia Disease of Protein Structure Sickle cell hemoglobin Hemoglobin S xxxxxx hemoglobin S less soluble when deoxygenated Filamentous hemoglobin causes sickling Single mutation Surface residue Glux Val6 in b subunit EV6 Normal erythrocytes Sickle cell E6V mutation Hydrophobic surface Hydrophobic surface pocket only in deoxygenated T state Left interface of two hemoglobin tetramers in mutant E6V Right Heme in red mutant Val in blue Book problems Questions 5 9 17 19 25 27 30 there are many good questions Feel free to work as many extra as you want Like 24 excellent question but long and wordy so didn t make the cut