I have occasionally seen in almost dried blood, placed between glass plates in a desiccator, rectangular crystalline structures, which under the microscope had sharp edges and were bright red. -Friedrich Ludwig Hunefeld, Der Chemismus in der thierischen Organisation, 1840 (one of the first observations of hemoglobin) Since the proteins participate in one way or another in all chemical processes in the living organism, one may expect highly significant information for biological chemistry from the elucidation of their structure and their transformations. -Emil Fischer, article in Berichte der deutschen chemischen Gesellschaft zu Berlin, 1906 Chapter 5 Protein Function *Knowing the 3-D structure of a protein is an important part of understanding how the protein functions
Ligand --- a molecule bound reversibly by a protein Binding site --- the site on protein to which a ligand binds Induced fit --- the structure adaptation that occurs between protein and ligand Substrate --- the molecule acted upon by enzymes Catalytic/active site --- the substrate/ligand binding site Noncatalytic functions of proteins
Heme: Reversible binding of a protein to a ligand: Oxygen-binding proteins protoporphyrin
The structure of myoglobulin *a single binding site for O 2 *78% helices (8) *His 93 or HisF8 (the 8 th residue in helix F) binds to heme *Bends between helices
Protein-ligand interactions can be described quantitatively P + L PL Ka = [PL]/[P][L] Kd = [P][L]/[PL] = (binding sites occupied)/(total binding sites) = [PL]/[PL] + [P] = [L]/([L] +1/Ka) Graphical representation of ligand binding = 0.5 [L] = 1/Ka, or Kd
Dissociation constant, K d = [P][L]/[PL] P + L PL Ka = [PL]/[P][L] = (binding sites occupied)/(total binding sites) = [PL]/[PL] + [P] = [L]/([L] +1/Ka) = [L]/([L] +Kd) When [L] = Kd = 0.5 (half saturation) [L] = 9 Kd = 0.9 Kd: the molar concentration of ligand at which half of the available ligand-binding sites are occupied Kd, affinity ( ? )
Table 5-1
When O 2 binds to Mb P 50 = 0.26 kPa = [L]/([L] +Kd) = [O 2 ]/([O 2 ] + Kd) = [O 2 ]/([O 2 ] + [O 2 ] 0.5 ) The concentration of a volatile substance in solution is always proportional to its partial pressure in the gas phase above the solution = pO 2 /(pO 2 + P 50 )
Protein structure affects how ligands bind 1. Steric effects 2. Molecular motions/breathing in the structure 1: 200 Binding ability O 2 :CO = 1: 20,000 (free heme)
Oxygen is transported in blood by hemoglobin (Hb) In arterial blood, Hb ~96% saturated In venous blood, Hb ~64% saturated P 50 = 0.26 kPa Mb Mb has only one subunit, as an oxygen-storage protein
Fig. 5-6 Comparison between Mb and Hb
Fig. 5-7 Comparison of aa between whale Mb and Hb , A-H helices Only 27 aa identical Pink: conversed in all known globins
Fig. 5-8 Dominant interactions between Hb subunits >30 aa 19 aa (hydrophobic, H-bonds, affected strongly upon O 2 binding)
Hb undergoes a structural change on binding oxygen Fig The T(tense) R(relaxed) transition O2O2 Max Perutz
Fig. 5-9 Some ion pairs that stabilize the T state of deoxyHb 22 11
Fig Changes in conformation near heme on O 2 binding
Hb binds oxygen cooperatively Fig A sigmoid (cooperative) binding curve pO 2 : 4 (in tissues) vs (in lungs) kPa Mb – a single subunit protein Hb – 4 subunits, an allosteric protein (96%) (64%)
Allosteric protein – a protein in which the binding of a ligand to one site affects the binding properties of another site on the same protein allos --- other stereos --- solid or shape Homotropic interaction --- liagnd = modulator Heterotropic interaction --- ligand = modulator O as both a normal ligand and an activating homotropic modulator for Hb
Fig Structure changes in a multisubunit protein undergoing cooperative binding to ligand.
Cooperative ligand binding can be described quantitatively Dissociation constant, Kd = [P][L] n /[PLn] P + nL PLn Ka = [PLn]/[P][L] n = (binding sites occupied)/(total binding sites) = [L] n /([L] n +Kd) = [L] n /Kd Log{ = n log [L] – log Kd (Hill equation, 1910) Log{ = n log pO 2 – log P 50 n H : the Hill coefficient (slope of Hill plot) When n H 1 ???? Fig. 5-14
Two models suggest mechanisms for cooperative binding Concerted (all-or-none), 1965 Sequential, 1966 Fig. 5-15
Hemoglobin also transports H + and CO 2 (from the tissues to the lungs and the kidneys) CO 2 + H 2 O H + + HCO 3 - HHb + + O 2 HbO 2 + H + Carbonic anhydrase in red blood cells Lungs vs. Tissues C + H 2 N- C- C- O O O R H H+H+ C - N- C- C- O-O- O O R H H Bohr effect (Christian Bohr, 1904) His 146 (His HC3) Amino-terminal residue Carbamino-terminal residue Binding of H + and CO 2 to Hb favors a transition to T state
KR
O 2 binding to Hb is regulated by 2,3-bisphosphoglycerate (BPG) HbBPG + O 2 HbO 2 +BPG 4 1 [BPG] during hypoxia C-O-P-O-C-O-P-O- H-C-HH-C-H - O - P=O O-O- C - = O - O H-H- = O - O-O- (highly abundant in erythrocytes) (8 mM at high altitudes, 5 mM at sea level) (heterotropic allosteric modulation) Hypoxia--lowered oxygenation of peripheral tissues
Fig Binding of BPG to deoxyHb T state T R O2O2 Positively charged aa ++ BPG is negatively charged
Sickle-cell anemia is a molecular disease of Hb Glu6 mutates to Val6 in two chains
Complementary interactions between proteins and ligands: The immune system and immunoglobulins MHC (major histocompatibility complex) all vertebrate cells macrophages, B cells
Over view of the immune response to a viral infection
Structure of a human class I MHC protein Recognized by T-cell receptor
The structure of immunoglobulin G (IgG)
Binding of IgG to an antigen
Induced fit in the binding of an antigen to IgG Heavy chain Light chain Kd~ M
The Ab-Ag interaction is the basis fro a variety of important analytical procedures Ployclonal vs. monoclonal Ab ELISA (enzyme-linked immunosorbent assay)
Immunoblot assay (Western Blot)
Protein interactions modulated by chemical energy Actin, myosin, and molecular motors Myosin S1
The major components of muscle
Structure of skeletal muscle relaxed contracted
Muscle contraction
Molecular mechanism of muscle contraction 3~4 pN of forces, 5~10 nm movement/cycle