Polypeptide Chains & Protein Structure

Chapter 4 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.

Peptide Bonds - General

• 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 is important for protein folding
• Peptide bonds are the name, independent of the amino acids being joined

Peptide Bonds - Polypeptide Main Chains

• Because of the conserved nature of peptide bonds, there is a repeating pattern within the main chain
• The main chain is the constant portion of the polypeptide but the side chains are variable
• Within the main chain there is a repeating pattern of NCCNCC

Peptide Bonds - Partial Double Bond Characteristic

• Rotation around the C-N peptide bond is restricted because of its partial double-bond characteristic
• The six atoms of the peptide group are rigid and planar as a consequence of the partial double bond characteristics

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
• Trans configuration is favored because 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

Proteins - Four Levels of Protein Structure

• Primary structure pertains to the linear sequence of amino acids
• Secondary structure pertains to localized interactions within a polypeptide
• Tertiary Structure pertains to the final folding pattern of a single polypeptide
• Quaternary Structure pertains to the folding pattern when multiple polypeptides are involved

Primary Structure - General

• Defines the linear arrangement of amino acids in a polypeptide
• The primary structure is presented from the N (amino) terminus to the C (carboxyl) terminus
• An example is: Tyr-Gly-Gly-Phe-Leu or YGGFL

Primary Structure:

• The information specifying correct folding is contained within the primary structure
• It is not yet possible 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
• It is maintained by hydrogen bonds between main-chain amide and carbonyl groups
• Examples include α-Helices and β-Sheets

Secondary Structure - Conserved Across Proteins

• Elements of secondary structure are found in different proteins
• They retain the same overall characteristics independent of protein context

Secondary Structure -Two Key Rules

• Viable forms of secondary structure must optimize the hydrogen bonding potential of main-chain carbonyl and amide goups
• Viable forms of secondary structure must 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
• There is an equal number of hydrogen bond donors and acceptors within the polypeptide main-chain
• This is important for optimizing hydrogen bonds

Secondary Structure - Conformation of the Polypeptide Chain

• 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) Ca-N and Psi (ψ) Ca-C
• Theoretically, phi and psi can each range from -180 to 180
• Steric interference prevents the formation of most conformations

Secondary Structure - Conformation of the Polypeptide Chain

• Ramachandran 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

α-Helix - Discovery

• In 1948 Linus Pauling spent a day sick in bed reading detective stories and began to doodle
• For this he received the Nobel Prize in Chemistry in 1954

α-Helix - Hydrogen Bonds

• Alpha (α) helix is a right-handed helix with 3.6 residues/turn
• It is stabilized by hydrogen bonds which run parallel to the axis of the helix
• Carbonyl groups point toward the C-terminus while amide groups point to the N-terminus
• Each carbonyl of residue n hydrogen bonds with the amide group with a residue of n+4

α-Helix - Amino Acid Sequence Affect Stability

• Most sequences can theoretically form an α-helix
• 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
• Each dipole communicated through a helix by hydrogen bonding gives the helix a net dipole
• The N terminus has a partial positive dipole charge and the C terminus has a partial negative dipole charge
• The dipole is stabilized by residues at each termini whose charge opposes the helix dipole
• Negatively charged residues (Asp, Glu) at the N terminus
• Positively charged residues (Lys, Arg, His) at the C terminus

α-Helix - Amphipathic Helicies

• Residues separated by three or four positions in the primary sequence will be on the same side of an α-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 phases

β Sheets - General

• ẞ Sheets involve multiple ẞ strands arranged side-by-side
• ẞ sheets are made up of ẞ strands
• ẞ Sheets often involve 4 or 5 strands
• Its conformation comes from fully extended polypeptide chains
• Its hydrogen Bonding Pattern is stabilized by hydrogen bonds between C=O and -NH on adjacent strands

β Sheets - Parallel and Anti-parallel

• Sheets are either parallel or anti-parallel
• In parallel sheets the strands run in the same direction
• In anti-parallel sheets the strands run in the opposite direction
• Anti-parallel sheets are more stable due to better geometry of hydrogen bonding

β Sheets - Mixed β-sheets

• sheets can be parallel, anti-parallel, or mixed
• Mixed sheets contain both parallel and antiparallel strands

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

Proteins - 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 because stability is defined as the tendency to maintain a native conformation
• Weak interactions predominate in stabilizing protein structure
• 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

Proteins - Folding

• Folded proteins occupy a low-energy state of the greatest stability, but this low-energy state may be only marginally stable
• Protein folding is a rapid process indicating that proteins don't sample all possible folding patterns
• Protein folding can be imagined as a funnel where a number of unstable conformations collapse to a single, stable folding pattern
• Some proteins spontaneously fold to their native conformation, others require the help of chaperones

Proteins - Denaturation

• Denaturation is the disruption of native conformation with loss of biological acitivty
• The 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

Proteins - Quaternary Structure

• Multiple subunits in which each subunit is a separate polypeptide
• It may involve multiple subunits of the same polypeptide of different polypeptides
• Subunits usually associate through non-covalent interactions
• Quaternary structure is usually reserved for proteins of more complex biological function

Proteins - Quaternary Structure

• The biological advantages of quaternary structure are
  • May help stabilize subunits and prolong a proteins life
  • 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 such as Hemoglobin
  • Conservation of a functional subunits is more efficient than selection for new protein with ideal function

Proteins - Structure and Function

• Biological roles are
  • Enzymes
  • Storage and transport
  • Physical cell support and shape
  • Mechanical movement
  • Decoding cell information
  • Hormones and or hormone receptors
  • Many specialized functions
• Diversity of function is enabled by diversity of structure
• Proteins show extreme structural and functional diversity

Proteins - Numbers and Diversity

• There is a different number of proteins depending on the organism
  • bacteria have ~ 5,000 proteins
  • fruit flies have ~ 16,000 proteins
  • humans have ~25,000 proteins
• This represents the minimum number of protein as addiontal isoforms are generated through post-translational modicfiation
• Humans may have up to a million different protein isoforms

Proteins - 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, which has an isoform that contains 34,350 amino acids
• The number of amino acids in a protein is approximated as dividing the protein molecular weight by 110 (average molecular weight of an amino acid)
  • For example, horse myoglobin has a molecular weight of 16,890
  • so 16,890/110 = 153.55 with the actual residues bein 153

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-convalent 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 are Keratin, Collagen, and Silk
• Globular Proteins are Myoglobin and 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
• At the level of secondar 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

Keratin - Coiled-Coils

• Two amphipathic helicies of keratin interact to bury their hydrophobic faces together
• 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

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 wil determine the strenth of the overall structure

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 which X is often proline
• It is a triplet repeat as (Gly-X-Y) (Gly-X-Y) (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
• The bulky side chains of proline are on the outside of the coiled-coil and the small side chains of the glycine residues are in the tightly packed core of the coiled-coil

Collagen - Post-translational Modifications

• The strenght of collagen arises from covalent linkages between the indiviual units into higher order structures
• Rather than disulfides, these linkages occur from residues that undergo post-transitional modification (hydroxyproline, hydroxylysine)
• More of these cross links occur with age, accounting for the increasing brittle character of aging connective tissue and tougher meat

Collagen - Post-translational Modifications

• The covalent crosslinks of collagen involve post-translationally modified residues for example hydroxyproline and hydroxylysine
• The enzymes performing these modifications requires Vitamin C
• Without these modified residues, collagen cannot form the stabilizing crosslinks
• Vitamin C deficiency (scurvy) leads to weekend structure of collagen which manifests in skin lesions, fragile blood vessels, bleeding gums etc

Collagen - Scurvy

• Magellan was first to sail around the globe but at the expense of 80% of his crew because of 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 Vitaman C
• 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
• Trails involving high doses of vitamin C showed no therapeutic value
• Instead inviduals taking the mega-does of vitams were more likely to develop cancer

Collagen - Genetic Diseases

• There are a number of genetic disorders involving collagen and related conncective tissues
• These include Osteogenisis imperfecta, Marfan's syndrome , Stickler symdrome and and Ehlers-Danlos Syndrome,
• These dieseases can be associated with brittle and abmornal bone structure, weekened cardiovascular capabilites, loose skin and joints and hyper-flexiblity

Collagen - Niccoló Paganini: Devil or Genetic Disorder?

• Niccolo Paganini is considered by many to be the greatest violin virtuosi to ever live
• Paganini was so beyond his peers that it was rumored that he had sold his soul to the devil
• Paganini was capable of palying htree octaves across four strings in a hand span, nearly an impossible feat
• Belived to have had Marfan's sundrome
• The resulting hyperextendbile joints allowed him to play music beyond the range of normal indivudals

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 structur, silk is primarily composed of beta sheets
• The fully extended polypeptides of of Beta offer considerable strength
• On a cross sectinal basis silk is one of the strongest known matrerials

Silk - Strength and Flexibility

• Appreciated the molecules basis on strength and flexibility of slik we need to cossdier its structure in each dimension
• Fully extended polypeptide chains (strength)
  • Association of strands by hydrogen bonding (flexible)
  • Assosiation 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 properites of silk are matched by challenges in its availability

Prions - A New Form of Infectious Disease

• Prion diseases are a novel paradigm infectious disease that are based on the misfolding of a self-protein into pathogenical and infectious conformations
• Prion diseases are fatal and untreatable neurodegenerative diseases

Prions - Disease-Specific Vaccines

• When a protein misfolds new region are exposed that antibody can bind to
• These misfolding-dependent epitopes are Disease-Specific Epitopes (DSEs)
• Disease specific epitopes (DSEs) appear ideal vaccine targets
• Anti-bodies induced against DSEs only bind the unhealthy form of the protein (PrPSc_ sparing the function of the heahtly dorm ( PrPC)

Prions - Other Infextious Proteins?

• Until recently, TSEs were a disinct categroy of neurodegenerative disoder exclusive in their defining characteristics of infextivity
• Inceasing evidence that the machanisms associated with porion self-propagation are consereved in other protien opathies
  • Alzheimers (beta-amyloid)
  • Parkinsons ( alpha-synuclein)
  • HUntington (huntingtin)
  • ALS (superoxide dismutase)

Explore phi ($\phi$) and psi ($\psi$) angles in polypeptide chains and Ramachandran plots. Learn about alpha ($\alpha$) helices, including their structure, stabilization, and amino acid composition. Explore protein subunit conservation.