Protein Structure

1. King Saud University College of Pharmacy Departments of Pharmaceutics PHT 560: Pharmaceutical Biotechnology Protein Structure Ibrahim A. Alsarra, Ph.D. Professor of Pharmaceutical Biotechnology

2. Outlines • Introduction • Protein Structure • Protein Folding • Protein Stability • Analytical Techniques

3. Introduction For a recombinant protein to become a human therapeutic, its biophysical and biochemical characteristics must be fully understood. The information must at least include: 1- Comparison of lot-to-lot reproducibility. 2- Establishment of allowable conditions for protein. 3- Identifying stability characteristics arising from long-term storage.

4. Protein Structure • Most proteins which are developed as therapeutic drugs perform specific functions by interaction with other small and large molecules, e.g. cell surface receptors, binding proteins, nucleic acid, carbohydrates and lipids. • The functional properties of protein are derived by their folding into three-dimensional structure. • Protein folding occurs based on polypeptide sequence in which twenty amino acids are connected through peptide bonds.

5. Protein Structure … cont. • All of the 20 amino acids consist of C carbon to which an amino group, a carboxyl group, a hydrogen and a side chain bind in L configuration. Structure of L-amino acids

6. Protein Structure … cont. • The amino acids are joined by condensation to yield a peptide bond consisting of a carboxyl group of an amino acid joined with the amino group of next amino acid. • The condensation gives an amide group, NH, at the N-terminal side of C and a carbonyl group, C=O, at the C-terminal side. These groups play an important role in protein folding. • These groups make a major energetic contribution to the formation of two important secondary structures, -helix and β-sheet Structure of peptide bond

7. Structures of 20 amino acids

10. Protein Structure … cont. Important facts:

11. Protein Structure … cont. Important facts: • At neutral pH, aspartic and glutamic acids are negatively charged and lysine and arginine are positively charged. • Histidine is positively charged at pH 7. • Tyrosine and cysteine are protonated and uncharged at neutral pH, but the become negatively charged above pH 10 and 8.

12. Protein Structure … cont. • Addition of methyl groups at position 1,3, and 7 of the natural substance xanthine produces the widely consumed compound caffeine. • Addition of methyl groups at position 1 and 3 or 3 and 7 produces the bronchodilator theophylline. Not all protein drugs and vaccines of the same name are identical

13. Xanthine

14. Protein Structure … cont. Not all protein drugs and vaccines of the same name are identical • The addition of hydroxy-methyl group to the antiherpes simplex drug acyclovir results in ganciclovir, which has anticytomegalovirus. • Cytomegalovirus retinitis, also known as CMV retinitis, is an inflammation of the eye's retina that can lead to blindness. Owl’s Eye

15. Acyclovir Ganciclovir

16. Protein Structure … cont. Secondary Structure: -Helix: • Immediately evident in the primary structure of protein is that each amino acid is linked by a peptide bond. • The amide, N=H, is a hydrogen donor and the carbonyl, C=O, is a hydrogen acceptor, and they can form a stable hydrogen bond when they are positioned in appropriate configuration of the polypeptide chain.

17. Protein Structure … cont. Secondary Structure: -Helix …cont.: • Such structures of the polypeptide chain are called secondary structure. • Two main structures, -helix and β-sheet, accommodate such stable hydrogen bonds. • Both ends of an -helix are highly polar.

18. Protein Structure … cont. Secondary Structure: -Helix …cont.: • The functional groups and hydrogen bonds are aligned in such way that would help the -helix to come in contact with side chains. These interactions called long range interaction actions, can stabilize the -helical structure and act as a folding unit. • Therefore, one side of the -helix is highly hydrophobic which will form an internal core, while the other side is relatively hydrophilic most likely exposed surface.

19. Protein Structure … cont. Secondary Structure: β-Sheet: • The β sheet (also β-pleated sheet or β strand) is a commonly occurring form of regular secondary structure in proteins. • typical strand is about five to ten amino acids long. • A β-sheet refers to an assembly of such strands that are hydrogen-bonded to each other. However, the term "β-sheet" is also sometimes used as a synonym of "β-strand", i.e., for a single segment of extended, hydrogen-bonded amino acids.

20. Protein Structure … cont. Secondary Structure: β-Sheet …cont: • β-strands can interact with each other in one of two ways either parallel or antiparallel evident in the primary structure of protein is that each amino acid is linked by a peptide bond.

21. Protein Structure … cont. Tertiary Structure: • Combination of the various secondary structures in a protein results in its three-dimensional structure. • Many proteins fold into a fairly compact, globular structure. • Folding a protein into a distinct three-dimensional structure determine its function. • Enzyme activity requires exact coordination of catalytically important residues in the three-dimensional space.

22. Protein Structure … cont. Forces: • Interactions occur between groups in proteins which are responsible for formation of their secondary, tertiary and quaternary structures. • Either repulsive or attractive interaction can occur between different groups. • Attractive interactions: hydrophobic interaction, hydrogen bonds, electrostatic interaction and van der Waals interactions. • The interactions are relatively weak and can be easily broken and formed, hence, folded protein structures arise from a fine balance between repulsive and attraction interactions.

23. Protein Structure … cont. Forces:H-Bonding: • In chemistry, a hydrogen bond is a type of attractive intermolecular force that exists between two partial electric charges of opposite polarity. Although stronger than most other intermolecular forces, the typical hydrogen bond is much weaker than both the ionic bond and the covalent bond.

24. Protein Structure … cont. Forces:H-Bonding …cont.: • As the name "hydrogen bond" implies, one part of the bond involves a hydrogen atom. The hydrogen atom must be attached to one of the elements oxygen, nitrogen or sulfur, all of which are strongly electronegative heteroatom. These bonding elements are known as the hydrogen-bond donor.

25. Protein Structure … cont. Forces: Hydrogen bonds in proteins and DNA: • Bonding between parts of the same macromolecule cause it to fold into a specific shape, which helps determine the molecule's physiological or biochemical role. • The double helical structure of DNA, for example, is due largely to hydrogen bonding between the base pairs, which link one complementary strand to the other and enable replication. • When two strands are joined by hydrogen bonds involving alternating residues on each participating strand, a beta sheet is formed. Hydrogen bonds also play a part in formingthe tertiary structure of protein through interaction of R-groups.

26. Protein Structure … cont. Hydration: • Water molecules are bound to proteins internally and externally. • The protein surface is extensive and consists of a mosaic of polar and non-polar amino acids, and it binds a large amount of water molecules, i.e. hydrated. • Water molecules can make significant contributions to protein stability. • Certain enzymes function in non aqueous solvent, provided that a small amount of water, just enough to cover the protein surface, is present. • Dried enzymes are in general inactive and become after they absorb certain amount of water per protein to give flexibility for function.

27. Protein Structure … cont. Protein Stability: • Proteins are neither chemically nor physically stable. • The protein surface is chemically highly heterogeneous and contains reactive groups, therefore, long term exposure of theses groups to environmental stresses causes various chemical alterations. • Alteration may include: hydrolysis,oxidation, aggregation, denaturation…etc.

28. Protein Structure … cont. Protein Stability • Physical stability of a protein is expressed as the difference in free energy, ∆Gu, between the native (N) and denatured (D) states. Thus, protein molecules are in equilibrium between the two states: • Protein stabilizing agents have been used to enhance storage stability of proteins. • Another approaches to enhances storages stability proteins is to lyophilize, or freeze-dry, the proteins.

29. Protein Structure … cont. Techniques for Characterizing Folding: • Conventional techniques used to obtain information of the folded structure of proteins are circulardischroism (CD), fluorescence, and Fourier transform infrared spectroscopies (FTIR). • CD and FTIR are widely used to estimate the secondary structure of proteins. (-helical content). • The β-sheet gives weak CD signals (variable in peak positions and intensities). However, FTIR can readily estimate the β-structure content as well as distinguish between parallel and antiparallel.

30. Protein Structure … cont. Techniques for Characterizing Folding: • None of these techniques can give the folded structure at the atomic level, i.e. they give no information on the exact location of each amino acid residue in the three dimensional structure of the protein. This is usually determined by NMR. • Hydrodynamic proprieties of proteins change greatly upon folding, going from elongated and expanded structures to compact globular ones.

31. Protein Structure … cont. Analytical Techniques : Blotting Techniques: • They are used to detect very low levels of unique molecules in milieu of proteins, nucleic acids and other cellular components. They can also detect aggregates or breakdown products occurring during long-term storage. • The method includes: 1- Transfer biomedical to a membrane (e.g. nitrocellulose). 2- Add specific reagents. 3- Filter the solution through a membrane which campsite biomedical. 4- Do fractionation if necessary.

32. Protein Structure … cont. Analytical Techniques : Blotting techniques include: • Southern Blots: used to detect DNA fragments. • Northern Blots: used to detect RNA fragments. • Western Blots: used to detect proteins by using labeled antibodies to detect specific proteins.

33. Protein Structure … cont. Analytical Techniques : Detection techniques include: • ELISA: (enzyme-linked immunosorbent assay). • Electrophoresis: gel or capillary. • Chromatography: size exclusion, reversed phase high performance liquid chromatography, hydrophobic interaction, ion exchange. • Bioassays. • Mass Spectrometry.

34. Protein Structure … cont. Detection techniques:ELISA: (Sandwich or Competitive) • The Enzyme-Linked Immuno Sorbent Assay is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. It uses two antibodies one of which is specific to the antigen and the other of which is coupled to an enzyme. This second antibody gives the assay its "enzyme-linked" name, and will cause a chromogenic or fluorogenic substrate to produce a signal.

35. Protein Structure … cont. Detection techniques:ELISA: • Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the Human Immunodeficiency Virus, HIV test or West Nile Virus) and also for detecting the presence of antigen. A 96-well microtiter plate such as the one shown above might be used for ELISA.

36. West Nile virus (or WNV) is a virus of the family Flaviviridae. It is found in both tropical and temperate regions. It mainly infects birds, but is known to infect humans, horses, dogs, cats, bats, chipmunks, skunks, squirrels, and domestic rabbits. The main route of human infection is through the bite of an infected mosquito. West Nile virus

37. W.N.V. has three different effects on humans. The first is an asymptomatic infection; the second is a mild febrile syndrome termed West Nile Fever; the third is a neuroinvasive disease termed West Nile meningitis or encephalitis. The second, febrile stage has an incubation period of 2 to 8 days followed by fever, headache, chills, diaphoresis (excessive sweating), weakness, lymphadenopathy (swollen lymph nodes), drowsiness, pain in the joints and symptoms like those of the common cold. West Nile virus… cont.

38. Occasionally there is a short-lived rash and some patients experience gastrointestinal symptoms including nausea, vomiting, loss of appetite, or diarrhea. All symptoms are resolved within 7 to 10 days, although fatigue can last for some weeks and lymphadenopathy can take up to two months to resolve. The more dangerous encephalitis is characterized by similar early symptoms but also a decreased level of consciousness, sometimes approaching near-coma. Deep tendon reflexes are hyperactive at first, later diminished. There are also extrapyramidal disorders. Recovery is marked by a long convalescence with fatigue. West Nile virus… cont.

39. Protein Structure … cont. Detection techniques:ELISA Method: • Apply a sample of known antigen to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. • The plate wells or other surface are then coated with serum samples of unknown antibody concentration, usually diluted in another species' serum. • The plate is washed, so that unbound antibody is removed. After this wash, only the antibody-antigen complexes remain attached to the well.

40. Protein Structure … cont. Detection techniques:ELISA Method: • The second antibodies are added to the wells, which will bind to any antigen-antibody complexes. These second antibodies are coupled to the substrate-modifying enzyme. • Wash the plate, so that excess unbound antibodies are removed. • Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorescent signal. • View/quantify the result using a spectrophotometer or other optical device.

41. Protein Structure … cont. Detection techniques:Capillary Electrophoresis • Capillary electrophoresis (CE) can be used to separate ionic species by their charge and frictional forces. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. Introduced in the 1960s, the technique of capillary electrophoresis (CE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte.

42. Protein Structure … cont. Detection techniques:Gel Electrophoresis • Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or iso-electric point. Gel electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

43. The isoelectric point (pI), sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). The pI is pH value at which the molecule carries no electrical charge or the negative and positive charges are equal. Isoelectric point

44. The pI value can affect the solubility of a molecule at a given pH. Such molecules have minimum solubility in water or salt solutions at the pH which corresponds to their pI and often precipitate out of solution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids which make up proteins may be positive, negative, neutral or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can thus be separated according to their isoelectric point (overall charge). Isoelectric point

45. DNA electrophoresis apparatus. An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates towards the camera.

46. Protein Structure … cont. Detection techniques:Chromatography • It is widely used in biotechnology not only in protein purification procedures, but also in assessing the integrity of the products. • Collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture which contains the analyte through a stationary phase, which separates it from other molecules in the mixture and allows it to be isolated using a mobile phase.

47. Protein Structure … cont. Detection techniques:Bioassay • Bioassay is a shorthand commonly used term for biological assay and is a type of in vitro experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism. Bioassays may be qualitative or quantitative, the latter often involving an estimation of the concentration or potency of a substance by measurement of the biological response that it produces.

48. Protein Structure … cont. Detection techniques:Bioassay • Quantitative bioassays are typically analyzed using the methods of biostatistics. Bioassays are essential in the development of new drugs. • In vitro bioassays can also measure changes in cell number or production of another protein factor in response to stimulation of cells by the protein therapeutics. The amount of the secondary protein produces can be estimated by using an ELISA.

49. Protein Structure … cont. Detection techniques:Bioassay • The use of bioassays include: • Measurement of the pharmacological activity of new or chemically undefined substances. • Investigation of the function of endogenous mediators. • Determination of the side-effect profile, including the degree of drug toxicity.

50. Protein Structure … cont. Detection techniques:Mass Spectrometry: • Mass spectrometry is an analytical technique used to measure the mass-to-chargeratio of ions. It is most generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample. • Molecular masses that differ from expected values indicate that the sample has been altered or the protein under investigation contains contaminants.