1. Chapter 6 Proteins in Action

2. 1. Hemoglobin is a multisubunit allosteric protein that carries O2 in erythrocyte. 1.1 Hemoglobin is a well-studied and well-understood protein. 1.1.1 It was one of the first proteins to have its molecular mass accurately determined. 1.1.2 The first protein to be characterized by ultracentrifuge. 1.1.3 The first protein to be associated with a specific physiological function.

3. 1.1.4 The first protein with a single amino acid substitution being related to a genetic disease (the beginning of molecular medicine). 1.1.5 The first multisubunit protein with its detailed atomic structure determined by X- ray crystallography. 1.1.6 The best understood allosteric protein.

4. 1.2 Determination of the atomic structure of hemoglobin A (from normal adult) is very revealing. 1.2.1 The protein molecule exists as a  2  2 tetramer. 1.2.2 Each subunit has a structure strikingly and unexpectedly similar to each other and to that of myoglobin, indicating quite different amino acid sequences can specify very similar 3-D structures. 1.2.3 Extensive interactions exist between the unlike subunits through noncovalent interactions. 1.2.4 Quaternary structure changes markedly when O 2 binds. Crystals of deoxyhemoglobin shatter (break) when exposed to O 2. 1.2.5 O 2 binds to the sixth coordination position of the ferrous iron (as in myoglobin).

5. 1.3 Hemoglobin is a much more intricate and sentient (sensitive) molecule than is myoglobin. 1.3.1 The oxygen-binding (dissociation) curve of hemoglobin is sigmoidal and that of myoglobin is hyperbolic. 1.3.2 Myoglobin has a higher affinity for O 2, evolved for O 2 storage. 1.3.3 Hemoglobin releases O 2 efficiently at low oxygen level tissues (evolved for O 2 delivery), myoglobin does not. 1.3.4 Oxygen binding of hemoglobin shows positive cooperativity. The binding of O 2 (the ligand) at one heme facilitates the binding of O 2 at the other hemes on the same tetramer (vice versa, unloading of oxygen at one heme facilitates the unloading of oxygen at the others). (Negative cooperativity refers to a decrease of activity.)

12. 1.3.5 Increasing concentrations of H+ (with a decrease of pH) or CO 2 lowers the O 2 affinity of hemoglobin (H+ and CO 2 has no effect on O 2 affinity of myoglobin). This is called Bohr effect, which helps the release of O 2 in the capillaries of actively metabolizing tissues. (melecular mechanism?) 1.3.6 One molecule of 2,3-diphosphoglycerate (BPG) binds to the central cavity of one tetramer of hemoglobin, which lowers its O 2 affinity. 1.3.7 Fetal hemoglobin (HbF) binds BPG less strongly than does hemoglobin A (adult) and consequently has a higher oxygen affinity. (physiological function? Extraction of O 2 from the mother)

13. 1.4 Cooperativity is a particular case of an allosteric effect 1.4.1 Allosteric effect refers to the phenomenon in which a molecule (allosteric effector) bound to one site on a protein causes a conformational change in the protein such that the activity of another site on the protein is altered (increased or decreased). 1.4.2 H+, CO 2, and BPG all show an allosteric effect (heterotrophic?) for the O 2 binding process of hemoglobin.

14. 1.5 Two models have been proposed to explain the allosteric regulation phenomena 1.5.1 The sequential model (proposed by Daniel Koshland, Jr.) hypothesizes that the binding of one ligand to one subunit changes the conformation of that particular subunit from the T state (with a low activity) to the R state (with a high acitvity), a transition that increases the activity of the other subunits for the ligand. 1.5.2 The sequential model can be analogized to the tearing process of postage stamps. (see fig.)

15. 1.5.3 The concerted model (proposed by Monod, Wyman, and Changeux) hypothesizes that symmetry is conserved in allosteric transitions (all subunits are in the same conformation) and the binding of each ligand increases the probability that all subunits in that molecule are converted to the R-state (with a high activity). All-or-none model. 1.5.4 The interplay between these different ligand-binding sites is mediated primarily by changes in quaternary structure. The contact region between two subunits can serve as a switch that transmits conformational changes from one subunit to another.

16. T: tense state, circle, less active; R: relaxed state, square, more active Concerted, all-or-noneSequential

17. 1.5.5 The functional characteristics of an allosteric protein are regulated by specific molecules in its environment. In another words, in the evolutionary transition from myoglobin to hemglobin, a macromolecule capable of perceiving information from its environment has emerged.

18. 1.6 Sickle-cell anemia was found to be caused by a single amino acid change in the  chain of hemoglobin molecules. 1.6.1 The hemoglobin molecule from sickle-cell anemia patients (HbS) was found to have a higher pI value (having more net positive charges). 1.6.2 Peptide fingerprinting (protease digestion + electrophoresis + chromatography) of HbS and HbA (wt) revealed that all but one of the peptide spots matched. 1.6.3 Amino acid sequencing revealed that HbS contains Val instead of Glu is at position 6 of the  chain! 1.6.4 The oxygen binding affinity and allosteric properties of hemoglobin are virtually unaffected by this change (the  6 is located at the surface of the protein).

19. 1.6.5 High concentration of deoxygenated HbS forms fiber participatates, which sickles the red blood cells, because the fiber formation is a highly concerted reaction. 1.6.6 Presence of Val6 on the  subunits generates a hydrophobic patch on the surface which complements with another hydrophobic patch formed only in deoxygenated HbS, thus generating the fiber precipitates (a polymer of HbS). 1.6.7 Sickle cell trait (heterozygote) confers a small but highly significant degree of protection against the most lethal form of malaria (probably by accelerating the destruction of infected erythrocytes, in Africa). 1.6.8 Fetal DNA can be analyzed for the presence of the HbS gene (prenatal DNA diagnosis).

20. 1.7 Thalassemias are genetic disorders characterized by defective synthesis of one or more hemoglobin chains. 1.7.1 This can be caused by a missing gene, impaired RNA synthesis or processing, generation of grossly abnormal proteins.

33. binding sites occupied [L]  = ------------------------------- = ------------- total binding sites [L] + K d  is a measurable quantity in experiment. [L] is the free ligand concentration.

36. The ordinate = n log[L] - log K d

38. Bohr’s effect and its molecular mechanism Release of O 2 in peripheral tissues Binding of O2 in lungs with release of H+.

39. Stabilization of T state??

42. legend???

44. Cavity in the tetramer hole

47. Mutation and molecular interaction’s changes

49. 2. Immunoglobulin superfamily members, found on cell surfaces or secreted, are widely used for specific molecular recognition. 2.1 An immunoglobulin G (IgG) molecule (of ~150 kD) was found to contain two light and two heavy chains, connecting to each other through disulfide bonds. 2.1.1 Papain digestion convert the IgG molecule into two Fab and one Fc fragments. 2.1.2 Each Fab fragment (containing one complete light chain and half of a single heavy chain) binds one molecule of antigen in a similar manner to the original immunoglobulin molecule.

50. 2.1.3 Each IgG molecule binds to two molecules of antigens (thus called bivalent). 2.2 Complete amino acid sequence analysis of purified myeloma patient’s immunoglobulins revealed strikingly that the L and H chains consist of variable and constant regions. 2.2.1 Residues 1 to 108 in the L chains are relatively variable, and 109 to 204 relatively constant. 2.2.2 Residues 1 to 108 in the H chains are variable, and 109 to 446 relatively constant. 2.2.3 Three segments in the L chain and three in the H chain display far more variability than do others, which are thus named as hypervariable segments (also called complementary-determining regions, or CDRs, because they determine antibody specificity).

51. 2.2.4 Amino acid sequence analysis revealed that the variable region of the light chain (VL) is similar in sequence to that of the heavy chain (VH). 2.2.5 The constant region of each heavy chain can divided into three parts (CH1, CH2, CH3) of similar sequences. 2.2.6 The amino acid sequence of the constant region of each light chain (CL) is similar to that of the three parts in the constant region of the heavy chains. Rodney Porter and Gerry Edelman were awarded the Nobel Prize in 1972 in Medicine or Physiology for their structure-function studies on the antibody molecules.

52. 2.3 X-ray crystallography studies revealed, strikingly, that the immunoglobulin molecules are made of 12 structurally similar domains. 2.3.1 Each domain has a recurring structural motif, called the immunoglobulin fold consisting of two broad sheets of antiparallel  -strands joined by a disulfide bond(?). 2.3.2 The CDRs of both VL and VH are located in loops at one end of the sandwich made of the two  - sheets that come together to form one antigen binding site. 2.3.3 The immunoglobulin core serves as a framework that allows almost indefinite variation of the CDR loops, corresponding to various antigen specificity of various antibodies.

53. 2.4 T cell receptors, Class I and Class II Major histocompatibility complex (MHC) proteins, and intracellular adhesion molecules (ICAMs) all contain domains similar to the immunoglobulin domains, thus belonging to the immunoglobulin superfamily. 2.4.1 The immunoglobulin domains (folds) are widely observed in these protein molecules, revealed by sequence homology (~20% identity) and similar foldings in some are confirmed by X-ray structure determination. 2.4.2 All these proteins are involved in molecular recognition (antigen recognition by antibodies, TCR, and MHC proteins; cell-cell interactions by ICAMs). 2.4.3 Most of the immunoglobulin superfamily members are found on cell surfaces (e.g., IgM, TCR, MHC) or secreted (IgG).

59. CDR1, CDR2, CDR3, CDR=complementarity determining region

70. 3. ATP-driven conformational cycles lead to muscle contraction. 3.1 Striated muscles contain overlapping arrays of thick and thin filaments. 3.1.1 The thick filaments are primarily made of myosin protein. 3.1.2 The thin filaments are primarily made of actin, tropomyosin, and the troponin complex. 3.1.3 Thick and thin filaments slide past each other in muscle contraction (a model proposed based on X-ray, light microscope, and EM studies, fig.)

71. 3.2 The force of muscle contraction arises from the interplay of myosin, actin, and ATP. 3.2.1 Myosin consists of two globular heads joined to a long  -helical coiled coil tail. 3.2.2 The myosin molecule can be cleaved by trypsin into two partially functional fragments, with one being able to form filaments, and the other being ATPase and able to bind actin. 3.2.3 Myosin molecules spontaneously assemble into filaments in solutions of physiological ionic strength and pH.

72. 3.2.4 Actin molecule exist in monomer form (G- actin) at low ionic strength and polymerizes into a fibrous form (F-actin, very similar to the thin filaments) at physiological (higher) ionic strength. 3.2.5 Threads of actomyosin complex are formed when mixing actin and myosin in solution. 3.2.6 Addition of ATP dissociates actomyosin into actin and myosin. 3.2.7 The actomyosin threads contracts when immersed in a solution containing ATP, K+, and Mg2+, whereas threads formed from myosin alone does not.

73. 3.2.8 Myosin, being an ATPase, can be regarded as an mechanoenzyme catalyzing the conversion of chemical bond energy into mechanical energy. 3.2.9 The ATPase activity of myosin is markedly enhanced when it binds to the polymerized form of actin (F-actin). 3.2.10 The hydrolysis of ATP drives the cyclic association and dissociation of actin and myosin. (model, fig.)

74. 3.3 The power stroke in contraction is driven by conformational changes in the myosin head. 3.3.1 In resting muscle, the myosin heads, with bound ADP and Pi, are unable to interact with the actin units in thin filaments because of steric interference by tropomyosin, a regulatory protein. 3.3.2 When muscle is stimulated, tropomyosin shifts position, and the myosin head (with bound ADP and Pi) reaches out from the thick filament and interact with the actin units on thin filaments. 3.3.3 The binding of myosin-ADP-Pi to actin leads to the release of ADP and Pi, which induces a major conformational change in the myosin head pulling the actin filament forward for about 100 Angstroms.

75. 3.3.4 Subsequently, ATP binds to the myosin head (myosin prefers binding to ATP than to actin) and thus detaches it from actin. 3.3.5 Finally, the bound ATP is hydrolyzed by the free myosin head, resetting it for the next interaction with the thin filament. 3.3.6 The essence of the process is a cyclic change both in the conformation of the myosin head and its affinity for actin. 3.3.7 The control of protein-protein interactions by bound nucleotides (ATP, GTP, etc) is a recurring theme in biochemistry.

87. 4. Troponin and tropomyosin mediate the regulation of muscle contraction by Ca2+. 4.1 Ca 2+ is released from sarcoplasmic reticulum (fig.) in muscle cells at the stimulation of a nerve impulse. 4.2 Ca 2+ controls muscle contraction by an allosteric mechanism (through conformational changes) in which the flow of information is in the following order: Ca 2+, the troponin complex, tropomyosin, actin, myosin. (are all their structures determined?)

88. 5. Other accessory proteins maintain the architecture on the myofibril and provide it with elasticity. 5.1 Springlike titin molecules, the largest protein so far found in nature (~3000 kD?), extend from the thick filaments to the Z disc. 5.2 Nebulin, another large protein molecule, is closely associated with the actin thin filaments, and consists of almost entirely of a repeating 35- amino-acid acting-binding motif.