Module 4: The Three-Dimensional Structure of Proteins PDF

Chapter 4: The Three-Dimensional Structure of Proteins Objectives: Characterize the nature of the peptide bond. Define the different levels of protein structure. Examine the characteristics of the different types of secondary structure. Examine the forces involved in protein folding...

Chapter 4: The Three-Dimensional Structure of Proteins Objectives: Characterize the nature of the peptide bond. Define the different levels of protein structure. Examine the characteristics of the different types of secondary structure. Examine the forces involved in protein folding and stability. Investigate the structure/function relationship of select proteins. Text Readings: Stryer 2nd or 3rd Edition All of Chapter 4  carboxyl group of acid and amino Peptide Bonds amino group of another amino acid. -General- ↑ water is molecule produced. Peptide bonds are covalent linkages between amino acids. Peptide bonds form by condensation reactions involving the loss of a water molecule. Formation of peptide bonds eliminates the α-carboxyl and α-amino charged groups, which will be important for protein folding. Peptide bonds are the same, independent of the amino acids being joined. "residues"  Peptide Bonds -Polypeptide Main Chains- As a result of the conserved nature of peptides bond, there is a repeating pattern within the main chain. The main chain is the constant portion of the polypeptide, the side chains are variable. ↳ everything EXCEPT the side chain. Within main-chain there is a repeating pattern of NCCNCC. amino Carboxy alpha  Peptide Bonds -Partial Double Bond Characteristic- Rotation around C-N peptide bond is restricted due to its partial double- bond characteristic. bond double be can ed. ↓ lose freedom of rotation. structural ↓ flexibility limits ways old to As a consequence of the partial double bond characteristic the six atoms of the peptide group are rigid and planar.  Peptide Bonds -Configuration- The partial double bond of the peptide bond creates cis-trans isomers. The oxygen of the carbonyl group and the hydrogen of the amide nitrogen are usually trans to each other. bonds peptide in to be tend trans · configuration EXCEPT proline cusually The trans configuration is favored as the cis configuration is more likely to cause steric interference between side chain groups. Steric exclusion means that two groups can’t occupy the same space at the same time. run into eachother  Proteins -Four Levels of Protein Structure- morepleasure Primary Structure is the linear sequence of amino acids. morepl on Secondary Structure is localized interactions within a polypeptide. Tertiary Structure is the final folding patter of a single polypeptide. Quaternary Structure is the folding pattern when multiple polypeptides are involved. -linear interactions nowtctfold mulesee  Primary Structure -General- Defines the linear arrangement of amino acids in a polypeptide. Primary structure is presented from the N (amino) terminus to the C (carboxyl) terminus. In this example: Tyr-Gly-Gly-Phe-Leu or YGGFL. free free carboxyl amino group group - END- BEGIN - -  Primary Structure -General- Primary Structure: The information specifying correct folding is contained within the primary structure. Is not yet possible (yet) to reliably predict three-dimensional structure based on primary structure. Primary structure is often determined through investigation of the corresponding gene.  Secondary Structure -General- Secondary Structure Represents localized patterns of folding in a polypeptide. Maintained by hydrogen bonds between main-chain amide and carbonyl groups. Examples include a-Helicies and b-Sheets. b-Sheet a-Helix  Secondary Structure -Conserved Across Proteins- Elements of secondary structure are found in different proteins. They retain the same overall characteristics independent of protein context. a-Helicies a-Helix b-Sheet b-Sheet  Secondary Structure -Two Key Rules- Viable forms of secondary structure must: Optimize the hydrogen bonding potential of main-chain carbonyl and amide groups. Represent a favored conformation of the polypeptide chain.  Secondary Structure -Main Chain Hydrogen Bonding Groups- Each peptide bond has a hydrogen bond donor and acceptor group. Equal number of hydrogen bond donors and acceptors within the polypeptide main-chain. This is important for optimizing hydrogen bonds. anything that each peptide bond CAN must form donor and acceptor = > - ,. H bond groups Donors Acceptors  Secondary Structure -Conformation of the Polypeptide Chain- has to be an allowable conformation Each α-carbon is held within the main-chain through single bonds, about which there is complete freedom of rotation. These bonds are defined as Phi (Φ) Cα-N and Psi (ψ) Cα-C. Theoretically, phi and psi can each range from –180 to 180. Steric interference prevents the formation of most conformations. c - C = psi c - N = phi = complete freedom Of rotation.  Secondary Structure -Conformation of the Polypeptide Chain- - quality control check. Ramachadran plots illustrate the possible combinations of phi and psi. Combinations of phi and psi that are actually observed in proteins are highlighted. These favored conformations correspond to the common elements of secondary structures. a b-Sheet favoured a-Helix  a-Helix > - first type of secondary -Discovery- structure e. In1948 Linus Pauling spent a day sick in bed reading detective stories. Bored he began to doodle….. For this he received the Nobel Prize in Chemistry in 1954.  α-Helix -Hydrogen Bonds- Alpha (α) Helix direction in which they - wrap around. Right-handed helix with 3.6 residues/turn. Stabilized by hydrogen bonds which run parallel to the axis of the helix. Carbonyl groups point toward the C-terminus; amide groups to the N-terminus. Each carbonyl of residue n hydrogen bonds with amide group of residue n+4. ↑ donors ↓ acceptors  α-Helix -Amino Acid Sequence Affect Stability- While most sequences can theoretically form an α-helix there are some guidelines and trends. > - cause redirection of polypeptide chain. Proline, because of its rigidity, is not usually found in α-helicies. Glycine, because of its flexibility, is also uncommon in α-helicies. Amino acids with side chain branches (Val, Thr, Ile) are less common due to steric interference. Amino acids with hydrogen bonding groups near the main-chain (Ser, Asp, Asn) are also less common. Charged residues tend to be positioned to form favorable ion pairs (residues of opposite charge separated by 3-4 positions).  α-Helix -The Helix Dipole- Every peptide bond has a small electrical dipole. N terminus Oxygen = nitrogen ① = Each dipole communicated through helix by hydrogen bonding giving the helix has a net dipole: N terminus has partial positive dipole charge longer nelix = C terminus has partial negative dipole charge. greaterare Dipole is stabilized by residues at each termini whose charge oppose the helix dipole. Negatively charged residues (Asp, Glu) at the N terminus Positively charged residues (Lys, Arg, His) at the C terminus C terminus  α-Helix -Amphipathic Helicies- BOTH polar & nonpolar face Residues separated by three or four positions in the primary sequence will be on the same side of an ⍺- helix. Residues separated by two residues in the primary structure will be on opposite sides of the helix. Positioning of hydrophobic and hydrophilic residues within the primary structure generates an amphipathic helix with polar and non-polar faces.  β Sheets - > discovered after alpha-sheets -General- Beta (β) sheets General: β Sheets involve multiple β strands arranged side-by-side individual β sheets are made up of β strands - > components withinsheets. β Sheets often involve 4 or 5 strands. Conformation : Fully extended polypeptide chains. Hydrogen Bonding Pattern: Stabilized by hydrogen bonds between C=O and -NH on adjacent strands.  β Sheets -Parallel and Anti-parallel- b Sheets are either parallel or anti-parallel In parallel b sheets the strands run in the same direction. In anti-parallel b sheets the strands run in opposite direction. Anti-parallel b sheets are more stable due to better geometry of hydrogen bonding. OPPOSITE DIRECTION SAME DIRECTION MORE BETTER ALIGNMENT: STABLE Parallel b sheets Anti-parallel b sheets  β Sheets -Mixed β-sheets- b sheets can be parallel, anti-parallel, or mixed. Mixed b sheets contain both parallel and antiparallel b strands. parallel enother anti-parallel eachother Mixed b sheet Mixed b sheet  β Sheets -Amphipathic β Sheets- Side chains tend to alternate above and below the polypeptide chain. Alternating polar and non-polar residues within the primary structure of a beta sheet will result in an amphipathic beta sheet. · lar non-polar face & face Non-polar Polar  Proteins -Tertiary Structure- Basic Facts about Tertiary Structure Tertiary structure represents the final folding pattern of a single polypeptide. The biological active folding pattern is the native conformation. Amino acid sequence determines tertiary structure. Tertiary structure describes the long range aspects of sequence interactions within a polypeptide. Residues separated by great distance in primary structure may be in close proximity in tertiary structure. Different proteins have different tertiary structures which relates to their unique functions. The tertiary structures of different proteins vary in their content of alpha helicies and beta sheets.  Proteins -Conformation is Stabilized by Weak Interactions- Proteins are only marginally stable (stability is defined as the tendency to maintain a native conformation). Weak interactions predominate in stabilizing protein structure. covalent interactions The protein conformation with the lowest free energy (the most stable) is usually the one with the maximum number of weak interactions. The stability of a protein reflects the difference in the free energies of the folded and unfolded states. a bility to unfold = a strength ↓ why ? manipulation of structure to dictate function.  Proteins -Folding- Folded proteins occupy a low-energy state of the greatest stability. This low-energy state may be only marginally stable. ↓ greatest amt Of Protein folding is a rapid process, indicating. disorder proteins don’t sample all possible folding patterns. Protein folding can be imagined as a funnel where a large number of unstable conformations collapse to a single, stable folding pattern. Some proteins spontaneously fold to their native conformations, others require the help of chaperones.  Proteins -Denaturation- Denaturation is the disruption of native conformation with loss of biological activity. energy required = small Energy required for denaturation is often small, perhaps only a few hydrogen bonds. Protein folding and denaturation is a cooperative process. For many proteins, denaturation is reversible. See when it starts to fall apart Man, it really falls apart Like boots or hearts when they start They really fall apart -Tragically Hip  Proteins -Quaternary Structure- Multiple subunits in which each subunit is a separate polypeptide. May involve multiple subunits of the same polypeptide or different polypeptides. Subunits usually associate through non-covalent interactions. Quaternary structure usually reserved for proteins of more complex biological function. SITE BINDING  Proteins -Quaternary Structure- There are a number of biological advantages associated with quaternary structure. May help stabilize subunits and prolong life of protein. Unique active sites produced at the interfaces between subunits. Help facilitate unique and dynamic combinations of structure/function through physiological changes in tertiary and quaternary structure (Hemoglobin). Conservation of functional subunits more efficient than selection for new protein with ideal function.  Proteins -Structure and Function- Biological roles of proteins include: denaturation = unfolding of Enzymes proteins. Storage and transport Physical cell support and shape most cell Mechanical movement action Decoding cell information = PROTEINS Hormones and/or hormone receptors Many other specialized functions Diversity of function enabled by diversity of structure. Proteins show extreme structural and functional diversity.  Proteins -Numbers and Diversity- Different organisms have different numbers of proteins. -bacteria have ~ 5,000 proteins -fruit flies have ~ 16,000 proteins -humans have ~25,000 proteins This represents the minimum number of proteins, additional isoforms are generated through post-translational modification. Humans may have up to a million different protein isoforms.  5) amino acids Proteins ↓ protein polypeptide- > -Size- Proteins are typically 100 to 1,000 amino acids in length. At 51 amino acids, insulin is often used as the threshold of when a polypeptide becomes a protein. The largest protein discovered to date is Titin, with an isoform containing 34,350 amino acids. The number of amino acids in a protein is approximated by dividing the proteins molecular weight by 110 (average molecular weight of an a.a). – For example, horse myoglobin has a molecular weight of 16,890. 16,890/110 = 153.55 (actual residues 153) – Estimate the molecular weight of titin.  Proteins -Five Important Facts- The function of a protein depends on its structure. The three dimensional structure of a protein is determined by its amino acid sequence. Non-covalent forces are the most important forces stabilizing protein structure. Within the huge number of unique protein structures, there are common structural patterns. An isolated protein usually exists in one, or a small number, of structural forms.  Proteins -Structure/Function Examples- Fibrous Proteins - > structural roles in the body. Keratin Collagen compare & Silk contrast Globular Proteins (next chapter) Myoglobin Hemoglobin  Keratin -Primary and Secondary Structure- Keratin is the principle component of hair, wool, horns, and nails. At the level of primary structure keratin contains a pseudo-seven repeat ↓ where positions a and d are hydrophobic residues. false ↓ not an absolute non-polar , not specific repeat (a b c d e f g ) (a b c d e f g ) (a b c d e f g ) (a b c d e f g ) At the level of secondary structure, keratin forms an alpha-helix. Residues from positions “a” and “d” end up on the same face of the helix resulting in a hydrophobic strip along the length of the helix. alterations :g in terms of structure a 4 - same face > - hydrophobi = non-polar  Keratin -Coiled-Coils- Two amphipathic helicies of keratin interact to bury their hydrophobic faces together. HYDROPHOBIC STRIP ALPHA HELICES RIGHT-HANDED = " COILED-cOlL" This results in the formation of a coiled-coil. Coiled-coils are formed when two or more helicies entwine to form a stable structure. The coiled-coil of keratin involves two right-handed helicies wrapping around each other in a left-handed fashion. GreaTeR STRENGTH =>  Keratin -Post-translational Stabilization- The strength of keratin arises from covalent linkages of individual units into higher-order structures. The individual units are linked together through disulfide bonds. The extent of disulfide bonding will determine the strength of the overall structure. COILED-COlL = building block ↓ bring many Together to form higherrvictures. ↓ DY forming covalentes on > by using cystine - residues ↓ disulfide linkages  Collagen -Primary and Secondary Structure- Collagen is a major protein of vertebrates (25% of total protein). At the level of primary structure, collagen contains repeats of Gly-X-Y where X is often proline. (Gly-X-Y) (Gly-X-Y) (Gly-X-Y) L triplet repeat Gly - X - Y At the level of secondary structure, collagen forms a left-handed helix of three residues per turn (as opposed to the 3.6 residues/turn of an α-helix).  Collagen -Coiled-Coils- Three left-handed helicies of collagen come together to form a coiled- coil. In collagen, three left-handed helicies wrap around each other in a right-handed fashion. 3 left handed helicies in a right handed fashion. The bulky side chains of proline are on the outside of the coiled-coil. The small side chains of the glycine residues are in the tightly packed core of the coiled- coil.  Collagen -Post-translational Modifications- The strength of collagen arise from covalent linkages between the individual units into higher order structures. Rather than disulfides, these linkages occur from residues that undergo post-translational modification (hydroxyproline, hydroxylysine). More of these cross links occur with age, accounting for the increasing brittle character of aging connective tissue and tougher meat. residues ↓ post-translation modification ↓ addhydrox) groups to proline & lysine = hydroxyproline hydroxylysine ↳ create covalent linkages  Collagen -Post-translational Modifications- The covalent crosslinks of collagen involve post-translationally modified residues (hydroxyproline, hydroxylysine). noascorbatea of no blo nation translaresidues tion ascorbate The enzymes performing these modifications requires Vitamin C. Without these modified residues, collagen cannot form the stabilizing crosslinks. Vitamin C deficiency (scurvy) leads to weakened structure of collagen which manifests in skin lesions, fragile blood vessels, bleeding gums, etc.  Collagen -Scurvy- Magellan was first to sail around the globe, at the expense of 80% of his crew to scurvy. Symptoms of scurvy include numerous bruises, tooth loss, poor wound healing, bone pain, and eventual heart failure. The demonstration that citrus prevents and cures scurvy was one of the first controlled human clinical trials. A study of 10% of University students don’t get enough vitamin C. https://en.wikipedia.or g/wiki/Scurvy Milder cases of scurvy cause fatigue, irritability, and susceptibility to illness.  Vitamin C -Too much of a good thing?- Later in life Linus Pauling (winner of two Nobel Prizes) proclaimed that high levels of Vitamin C could help avoid colds, cure cancer, and prolong life. Trials involving high doses of Vitamin C showed n0 therapeutic value. Instead, individuals taking the mega-doses of vitamins were more likely to develop cancer. made cancer > - worse. severity https://upload.wikimedia.org/ https://www.goodreads.co wikipedia/en/a/ac/Vitamin_C_ m/book/show/685175.Can and_the_Common_Cold_%28 cer_and_Vitamin_C book%29.jpg  Collagen -Genetic Diseases There are a number of genetic disorders involving collagen and related connective tissues. These include Osteogenesis imperfecta, Marfan’s syndrome, Stickler syndrome, and Ehlers-Danlos syndrome. These diseases can be associated with brittle and abnormal bone structure, weakened cardiovascular capabilities, loose skin and joints, and hyper-flexibility. advantages & disadvantages. https://en.wikipedia.org/wiki/Ehlers– Danlos_syndromes#/media/File:Ehlers-Danlos_thumb.jpg  Collagen -Niccolό Paganini: Devil or Genetic Disorder?- Niccolò Paganini is considered by many the greatest violin virtuosi to have ever lived. Paganini was so beyond his peers that it was rumored that he had sold his soul to the devil. Paganini was capable of playing three octaves across four strings in a hand span, a nearly impossible feat. Believed to have had Marfan’s syndrome. The resulting hyper-extendible joints https://upload.wikimedia.org/wikipedia/co allowed him to play music beyond the mmons/f/f4/Nicolo_Paganini_by_Richard_ James_Lane.jpg range of “normal” individuals. https://en.wikipedia.org/wi ki/Marfan_syndrome#/med ia/File:Marfan_thumb_sign.png  Silk -Primary and Secondary Structure- Silk fibroin is produced by insects and spiders for formation of webs and cocoons. Webs and cocoons need to be both strength and flexibility. At the level of primary structure, most silk has a six residue repeat. (GSGAGA) (GSGAGA) (GSGAGA) At the level of secondary structure, silk is composed primary from beta- sheets. The fully extended polypeptides of b offer considerable strength. On a cross sectional basis silk is one of the strongest known materials.  Silk -Strength and Flexibility- To appreciate the molecular basis of the strength and flexibility of silk, one needs to consider its structure in each dimension. Fully extended polypeptide chains (strength). Association of strands by hydrogen bonding (flexible). Association of sheets by van der Waals and hydrophobic interactions (flexible).  Silk -Medical Applications and Genetic Engineering- Due to its enticing properties, spider silk has enormous potential for medical applications. The exciting properties of silk are matched by challenges of its availability. https://sites.google.com/site/wwwgmoinf ocom/home/spider-goats  Prions -A New Form of Infectious Disease- Prion diseases are a novel paradigm of infectious disease based on the misfolding of a self-protein into a pathological, infectious conformation. Prion diseases are fatal, untreatable neurodegenerative diseases. PrPC PrPSc (Healthy (Unhealthy Conformation) Conformation) https://www.telegraph.co.uk/news/uknews/1565922/ Gummer-friend-dies-of-mad-cow-disease.html  Prions -Disease-Specific Vaccines- When a protein misfolds, new regions are exposed for antibody binding. These misfolding-dependent epitopes are termed Disease-Specific Epitopes (DSEs). Disease specific epitopes (DSEs) appear ideal vaccine targets. Antibodies induced against DSEs only bind the unhealthy form of the protein (PrPSc) sparing the function of the healthy form (PrPC). Effect of conformation-specific immunotherapy on endogenous PrPC and PrPSc.  Prions -The Only Infectious Proteins?- Until recently, TSEs were a distinct category of neurodegenerative disorder, exclusive in their defining characteristic of infectivity. Increasing evidence that the mechanisms associated with prion self- propagation are conserved in other proteinopathies. – Alzheimers (β-amyloid) – Parkinsons (α-synuclein) – Huntingtons (huntingtin) – ALS (superoxide dismutase)  Copyright Sourcing Images/Figures/Tables from Textbook – Permission: Courtesy of MacMillan Learning. Slide 6: Source: Lehninger Principles of Biochemistry (2000) 3rd Edition, page 129. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 15a: Source: http://paulingblog.wordpress.com/2011/03/09/the-alpha-helix/ Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act. Slide 15b: Source: http://richtervideos.com/LinusPaulingCrusadingScientist/ Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act. Slide 18a: Source: Lehninger Principles of Biochemistry (2012) 6th Edition, page 118. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 18b: Source: Lehninger Principles of Biochemistry (2008) 5th Edition, page 120. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 19a: Source: http://guweb2.gonzaga.edu/faculty/cronk/CHEM440pub/L09.html Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act. Slide 19b: Source: https://commons.wikimedia.org/wiki/File:Amphipathic_Alpha_Helix.png Permission: CC BY-SA 4.0 Courtesy of Linnikh. Slide 23: Source: Lehninger Principles of Biochemistry (2008) 5th Edition, page 120. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 27: Source: Lehninger Principles of Biochemistry (2008) 5th Edition, page 141. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 35: Source: http://guweb2.gonzaga.edu/faculty/cronk/CHEM440pub/L09.html Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act. Slide 36: Source: http://guweb2.gonzaga.edu/faculty/cronk/CHEM440pub/L09.html Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act.  Copyright Sourcing Slide 37: Source: Lehninger Principles of Biochemistry (2008) 5th Edition, page 124. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 40: Source: Lehninger Principles of Biochemistry (2008) 5th Edition, page 125. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 42: Source: https://en.wikipedia.org/wiki/Scurvy#/media/File:JamesLind.jpg Permission: Public Domain. 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Slide 49a: Permission: Courtesy of course author Scott Napper, Department of Biochemistry, Microbiology & Immunology, University of Saskatchewan. Slide 49b: Source: https://www.telegraph.co.uk/news/uknews/1565922/Gummer-friend-dies-of-mad-cow-disease.html Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act. Slide 50: Permission: Courtesy of course author Scott Napper, Department of Biochemistry, Microbiology & Immunology, University of Saskatchewan. Slide 51: Source: http://science.sciencemag.org/content/sci/326/5958/1337.full.pdf Permission: This material has been reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act.