BCH 5045 Graduate Survey of Biochemistryhort.ifas.ufl.edu/faculty/guy/bch5045/Lecture Files/Lecture 9.pdf · CHAPTER 12. Biosignaling. ... Moreover, in a clinical study of 12 patients

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BCH 5045

Graduate Survey of Biochemistry

Instructor: Charles GuyProducer: Ron Thomas

Director: Marsha Durosier

Lecture 9Slide sets available at:

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• LEHNINGER• PRINCIPLES OF BIOCHEMISTRY

• Fifth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 12

Biosignaling



Nitric oxide (NO) is a gaseous signaling molecule and neurotransmitter. Its role in biology was established in 1987 by Palmer et al. NO acts as an endothelium-derived relaxing factor that mediates the relaxation of arterial wall smooth muscle (SM) causing vasodilation. The EDRF (NO) is released in response to mechanical force and neurohumoral mediators acetylcholine, bradykinin and histamine.

NO stimulates muscle guanylate cyclase activity leading to an increase in cGMP and muscle relaxation. cGMP-induced SM relaxation is mediated mainly by cGMP-dependent protein kinase activation.

Interestingly, free radicals of NO act as a neurotransmitter in nonadrenergic noncholinergic nerves in the peripheral nervous system. NO exists in several oxidation-reduction states: a reduced form NO•, correctly termed nitric oxide; NO+, a nitrosonium ion; and finally NO or nitric monoxide.



NO signaling involves several molecular events culminating in a reduction in intracellular Ca2+ concentration and a decrease in the sensitivity of the contractile system to Ca2+. NO is also a neurotransmitter involved in intestinal relaxation in peristalsis and is also important in penile erection.

It is considered to be involved in various effects of the neurotransmitter glutamate acting at NMDA receptors in the brain. NO is synthesized from L-arginine in neurons and other cells by the enzyme nitric oxide synthase.



Palmer RM, Ferrige AG, Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524-526.Endothelium-derived relaxing factor (EDRF) is a labile humoral agent, which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. NO was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.



Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, Osterloh IH, Gingell C (1996) Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 8: 47-52.Sildenafil (Viagra, UK-92,480) is a novel oral agent under development for the treatment of penile erectile dysfunction. Erection is dependent on nitric oxide and its second messenger, cyclic guanosine monophosphate (cGMP). However, the relative importance of phosphodiesterase (PDE) isozymes is not clear. We have identified both cGMP- and cyclic adenosine monophosphate-specific phosphodiesterases (PDEs) in human corpora cavernosa in vitro. The main PDE activity in this tissue was due to PDE5, with PDE2 and 3 also identified. Sildenafil is a selective inhibitor of PDE5 with a mean IC50 of 3.9 nM. In human volunteers, we have shown sildenafil to have suitable pharmacokinetic and pharmacodynamic properties (rapid absorption, relatively short half-life, no significant effect on heart rate and blood pressure) for an oral agent to be taken, as required, prior to sexual activity. Moreover, in a clinical study of 12 patients with erectile dysfunction without an established organic cause, we have shown sildenafil to enhance the erectile response (duration and rigidity of erection) to visual sexual stimulation, thus highlighting the important role of PDE5 in human penile erection. Sildenafil holds promise as a new effective oral treatment for penile erectile dysfunction.



Because the NO-cGMP signaling pathway is a component of several biological processes, compounds that specifically influence the pathway have potentially important therapeutic uses and significant economic value. I am sure most everyone will recognize the popular name of at least one such compound. So biochemically what exactly does sildenafil do? http://www.bing.com/videos/search?q=viva+viagra+commerical+&view=detail&mid=7AF10917CFD454D98ABD7AF10917CFD454D98ABD&first=0&FORM=LKVR2

Can you figure out how to make NO from L-arginine?“Viagra is among the company’s top 10 best-selling drugs, behind such products as Lipitor, Zoloft and Celebrex. Viagra accounted for $1.87 billion in global sales last year, including $1.1 billion in sales to American men.”

Excerpt from: MSNBC“Pfizer ordered to pull Viagra commercials”

Associated Press; Updated: 9:22 a.m. ET Nov 16, 2004

http://www.bing.com/videos/search?q=viva+viagra+commerical+&view=detail&mid=7AF10917CFD454D98ABD7AF10917CFD454D98ABD&first=0&FORM=LKVR2�

http://www.bing.com/videos/search?q=viva+viagra+commerical+&view=detail&mid=7AF10917CFD454D98ABD7AF10917CFD454D98ABD&first=0&FORM=LKVR2�



Image provided by H. Criss Hartzell with permission. From H. CrissHartzell, “The Stress of Relaxation” Science 317: 1331-1332. 7 September 2007



Figure 1. NO-cGMP signaling pathway. From : Bivalacqua TJ, Champion HC, Hellstrom WJ, Kadowitz PJ (2000) Trends Pharmacol Sci 21: 484-489



This slide is not in the lecture video

NO chemistry. NO is a free radical and while free radicals are, by definition, generally unstable and highly reactive, however NO is rather chemically stable. NO does react readily with other free radicals such as O2̄̇ (superoxide) or lipid peroxyl radicals.

NO does form stable high affinity bonds with ferrous hemes and non-heme iron which is necessary for the activation of the guanylatecyclase activity.

Overall, NO is uncharged and is surprisingly perhaps, highly soluble in hydrophobic environments and this allows for its free diffusion across biological membranes. Its small size and solubility in nonpolar environments allows it to serve as a rapidly diffusible signal distant from its site of synthesis. The half-life for NO in some tissues is estimated to be in the range of 0.002 s to longer than 2 s.



• LEHNINGER• PRINCIPLES OF BIOCHEMISTRY

• Fifth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 4

The Three-Dimensional Structure of Proteins



This is a depiction of folding from a random chain to secondary and tertiary structure



Presenter

Presentation Notes

FIGURE 4-1 Structure of the enzyme chymotrypsin, a globular protein. A molecule of glycine (blue) is shown for size comparison. The known three-dimensional structures of proteins are archived in the Protein Data Bank, or PDB (see Box 4-4). The image shown here was made using data from the PDB entry 6GCH.



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FIGURE 4-2a The planar peptide group. (a) Each peptide bond has some double-bond character due to resonance and cannot rotate.



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FIGURE 4-2b The planar peptide group. (b) Three bonds separate sequential α carbons in a polypeptide chain. The N—Cα and Cα—C bonds can rotate, described by dihedral angles designated Φ and Ψ, respectively. The peptide C—N bond is not free to rotate. Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups. FIGURE 4-2d The planar peptide group. (d) By convention, Φ and Ψ are 180° (or –180°) when the first and fourth atoms are farthest apart and the peptide is fully extended. As the viewer looks out along the bond undergoing rotation (from either direction) the Φ and Ψ angles increase as the fourth atom rotates clockwise relative to the first. In a protein, some of the conformations shown here (e.g., 0°) are prohibited by steric overlap of atoms. In (b) through (d), the balls representing atoms are smaller than the van der Waal’s radii for this scale. FIGURE 4-3 Ramachandran plot for L-Ala residues. Peptide conformations are defined by the values of Φ and Ψ. Conformations deemed possible are those that involve little or no steric interference, based on calculations using known van der Waals radii and dihedral angles. The areas shaded dark blue represent conformations that involve no steric overlap and thus are fully allowed; medium blue indicates conformations allowed at the extreme limits for unfavorable atomic contacts; the lightest blue indicates conformations that are permissible if a little flexibility is allowed in the dihedral angles. The yellow regions are conformations that are not allowed. The asymmetry of the plot results from the L stereochemistry of the amino acid residues. The plots for other L residues with unbranched side chains are nearly identical. Allowed ranges for branched residues such as Val, Ile, and Thr are somewhat smaller than for Ala. The Gly residue, which is less sterically hindered, has a much broader range of allowed conformations. The range for Pro residues is greatly restricted because Φ is limited by the cyclic side chain to the range of –35° to –85°.



In 1951, Pauling and Corey published seven papers back-to-back in PNAS on the atomic coordinates for two helical configurations of polypeptides, the pleated sheet structure, the structure of keratin in a feather rachis, the structure of hair and muscle and collagen fibers and the polypeptide configurations in hemoglobin and other globular proteins that would rock the biochemistry world. And, without question their work has provided an important key to understanding the functions of proteins at the molecular level. Herman Branson was an early collaborator with Pauling and Corey and helped them to decipher and verify protein structures. Pauling and Corey were an original odd couple, or a perfect case where opposites attract, a paradigm well known in biochemistry. Pauling was a showman and loved to entertain and charm scientific audiences, but Corey disliked social events and did not like to attract attention to himself. Their collaboration would last almost three decades. Pauling and Corey used x-ray crystallography of short peptides from several proteins to elucidate the alpha helix and beta sheet structures of proteins. Pauling would receive the 1954 Nobel Prize in Chemistry for his work on protein structure.



The alpha helix shown in ball and stick form and space filling models. If the space fill model (d) were to be rotated to an end on view so that we could look right down the center of the helical axis like in (c) we would see no open space. All the space would take up by the van der Waals radii of the atom electron clouds. What exactly does the formation of an alpha helix represent in terms of the energy status of the helical structure? Does it represent an energy maximum or energy minimum? Hint, what does structural stability reflect?

Note intrahelicalhydrogen bonding

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Presentation Notes

FIGURE 4-4 Models of the α helix, showing different aspects of its structure. (a) Ball-and-stick model showing the intrachain hydrogen bonds. The repeat unit is a single turn of the helix, 3.6 residues. (b) The α helix viewed from one end, looking down the longitudinal axis (derived from PDB ID 4TNC). Note the positions of the R groups, represented by purple spheres. This ball-and-stick model, which emphasizes the helical arrangement, gives the false impression that the helix is hollow, because the balls do not represent the van der Waals radii of the individual atoms. (c) As this space-filling model shows, the atoms in the center of the α helix are in very close contact. (d) Helical wheel projection of an α helix. This representation can be colored to identify surfaces with particular properties. The yellow residues, for example, could be hydrophobic and conform to an interface between the helix shown here and another part of the same or another polypeptide. The red and blue residues illustrate the potential for interaction of negatively and positively charged side chains separated by two residues in the helix.



The rotational direction of a helix can be determined you the use of one’s hands. If going from N-terminus to C-terminus starting with N-terminus pointing down, if the helix pattern follows your right hand, then it is a right-handed helix. Similarly if the rotational direction follows that of your left hand, then the helix is a left-handed helix.

Because the amino-terminus carries a positive charge and the carboxyl-terminus has a negative charge and there is a retention of this orientation for the partial positive and negatives charges of the hydrogen bonds that form along the helical chain, it is state that the alpha helix is a macro-dipole.

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Presentation Notes

FIGURE 4-5 Helix dipole. The electric dipole of a peptide bond (see Figure 4-2a) is transmitted along an α-helical segment through the intrachain hydrogen bonds, resulting in an overall helix dipole. In this illustration, the amino and carbonyl constituents of each peptide bond are indicated by + and – symbols, respectively. Non-hydrogen-bonded amino and carbonyl constituents of the peptide bonds near each end of the α-helical region are shown in red.



Beta pleated sheets occur in two forms, antiparallel and parallel referring to the direction the polypeptide chains are oriented. Either way, the arrangement of a the amino and carboxyl groups are spaced in a way that allows for the formation of the interchain hydrogen bonding. Can this be helpful for protein structure stability? If so, then how? Clearly the peptide bonds in the beta-structure have different φ and ψbond angles than the alpha helix.

Note the inter chain hydrogen bonding.

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FIGURE 4-6a The β conformation of polypeptide chains. These top and side views reveal the R groups extending out from the β sheet and emphasize the pleated shape described by the planes of the peptide bonds. (An alternative name for this structure is β-pleated sheet.) Hydrogen-bond cross-links between adjacent chains are also shown. The amino-terminal to carboxyl-terminal orientations of adjacent chains (arrows) can be the same or opposite, forming (a) an antiparallel β sheet or (b) a parallel β sheet.



Approximately one-third of amino acids in a globular protein can be associated with loops and turns. A common type of turn in proteins is the beta turn that links two regions of beta structure. The turn is usually four amino acids in length and the orientation of the residues are such that a hydrogen bond can form between and first and four amino acids. Proline is often a constituent of beta turns and can occur in the cis instead of the trans conformation typical of virtually all peptide bonds.

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FIGURE 4-7a Structures of β turns. (a) Type I and type II β turns are most common; type I turns occur more than twice as frequently as type II. Type II β turns usually have Gly as the third residue. Note the hydrogen bond between the peptide groups of the first and fourth residues of the bends. (Individual amino acid residues are framed by large blue circles.) FIGURE 4-7b Structures of β turns. (b) Trans and cis isomers of a peptide bond involving the imino nitrogen of proline. Of the peptide bonds between amino acid residues other than Pro, more than 99.95% are in the trans configuration. For peptide bonds involving the imino nitrogen of proline, however, about 6% are in the cis configuration; many of these occur at β turns.



Ramachandran plots show the range of φ and ψ bond angles in proteins. A survey of many proteins whose 3-dimensional structures are known, shows that the φ and ψbond angles fall into discrete regions of the bond angle universe. This should not be surprising as different structural forms depend on the arrangement of atoms around the peptide bonds. Then it follows that the different amino acids will favor or disfavor certain types of structures.

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FIGURE 4-8a Ramachandran plots showing a variety of structures. (a) The values of Φ and Ψ for various allowed secondary structures are overlaid on the plot from Figure 4-3. Although left-handed α helices extending over several amino acid residues are theoretically possible, they have not been observed in proteins.



Hair is composed of alpha Keratin a right-handed alpha helix. See how the organization of the chains, protofilament and protofibril contribute to the structure of the hair shaft. Although it may not seem like it, but this arrangement of keratin chains is designed for strength. Note that the protofilaments overlap in the protofibril. In building construction, we employ the same principles to create strength and stability. The organization of the hair shaft is an example of the quaternary structural organization of proteins.

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FIGURE 4-10a Structure of hair. (a) Hair α-keratin is an elongated α helix with somewhat thicker elements near the amino and carboxyl termini. Pairs of these helices are interwound in a left-handed sense to form two-chain coiled coils. These then combine in higher-order structures called protofilaments and protofibrils. About four protofibrils—32 strands of α-keratin in all—combine to form an intermediate filament. The individual two-chain coiled coils in the various substructures also seem to be interwound, but the handedness of the interwinding and other structural details are unknown. FIGURE 4-10b Structure of hair. (b) A hair is an array of many α-keratin filaments, made up of the substructures shown in (a).

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BCH 5045 Graduate Survey of Biochemistryhort.ifas.ufl.edu/faculty/guy/bch5045/Lecture Files/Lecture 9.pdf · CHAPTER 12. Biosignaling. ... Moreover, in a clinical study of 12 patients