Proteins: Secondary Structure and Fibrous Proteins

• 4 levels of protein structure (primary, secondary, tertiary, quaternary)
  
  
• Partial double bond character of peptide bond has important consequences for protein structure.
  
  
• major types of secondary structure found in many proteins:
  • a helix
  • b conformation
  • (b turns)
    
    
• Fibrous proteins demonstrate secondary structural motifs assembled into supersecondary structures.
  • a keratins
  • collagen
  • silk fibroin
    

Levels of Protein Structure

• Proteins' functions can only be understood in terms of their structures. 
• 3-dimensional structures of many proteins have been determined, from which a few general principles can be derived.
  
• Different aspects of protein structure: 4 levels of protein structure:
  • Primary Structure = the amino acid sequence of its polypeptide chain(s) + locations of any disulfide bonds in the sequence.
    • Every protein has a unique amino acid sequence.
    • 1^o structure is stabilized/held together by covalent bonds (peptide bonds)
      
  • Secondary Structure = spatial arrangement of polypeptide backbone, ignoring conformations of sidechains
    • recurring structural patterns, e.g. a-helix and b-conformation
    • mainly stabilized by hydrogen bonds
  • Tertiary Structure = complete three dimensional structure of entire polypeptide, including conformations of side chains
    • Sometimes the same folding patterns within the 3^o structure are seen in many proteins, so motifs (general "folds") have names, e.g. a/b barrel, 4-helix bundle, etc.
    • stabilized by all 4 types of noncovalent interactions, and sometimes in extracellular proteins by disulfide bonds as well (covalent)
  • Quaternary Structure = the number and spatial arrangement of subunits (individual polypeptide chains) in 3-dimensional structures of proteins that are composed of 2 or more polypeptide chains.
    • stabilized by all 4 types of noncovalent interactions, and sometimes in extracellular proteins by disulfide bonds as well (covalent)

Fig. 5-16 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): Levels of protein structure

Secondary Structure

• partial double bond character of peptide bond
• Fig. 6-2a (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.) The planar peptide group

• 3 CONSEQUENCES of partial double bond character of peptide bond:
  • cis-trans isomerism
    • In proteins, aC atoms of adjacent residues are almost always in trans configuration (otherwise there can be steric hindrance)
  • planarity of peptide group
    • OCNH atoms of polypeptide backbone are planar
    • aC atoms on either side of peptide bond are coplanar with the 4 peptide bond atoms
    • There are thus 6 atoms in the same plane: C_a1-CO-NH-C_a2. 
  • torsion angles:  phi (f) and psi (y) (also called dihedral angles, or angles of rotation)
• Figs. 6-2b and 6-2c (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.):

The only degrees of freedom for rotation in the polypeptide backbone are around the bonds to the Ca carbon: phi (f) or psi (y). Each residue in 3-dimensional structure of protein has its own angular coordinates (f,y). All together, entire set of (f,y) angles for a polypeptide chain define the whole course of folding of backbone of chain in space. However, there are significant limitations as to which angles of f and y can be used, due to steric clashes between atoms.  Click (here) to see an animation showing steric clashes as the elements of the peptide backbone rotate about f or y.

• Small residues like L-Ala have more "permitted" backbone conformations than larger residues like L-Trp. (Gly has by far the most conformational freedom.)
• Pro residues have a limited range of f angle (f between about -35^o and -85^o)
  • Restriction in f is due to the cyclic structure involving the a-imino N.
  • Cyclic structure generates a natural "elbow" in polypeptide chain backbone.
    

• A Ramachandran Plot shows those regions of (f,y) where there are no steric conflicts.
  left side: Fig. 6-3. (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.) Ramachandran plot for L-Ala residues.
  right side: (f,y) angles for backbone conformations of common secondary structures (figure drawn by Dr. Michael Wells)

• For proteins, there are two major regions of (f,y) space where the coordinates of many residues are found:
  • b-strand conformation
  • a-helix conformation
• Repetition of same angular coordinates for successive residues gives rise to recognizable, repetitive patterns for polypeptide backbone folding: secondary structures

Secondary Structural Elements

• IMPORTANT PRINCIPLE IN PROTEIN FOLDING:
  • The very common secondary structures maximize the number of intra-protein hydrogen bonds involving the polar backbone groups of the peptide bonds, the amide N-H and C=O.  WHY?  (Keep that question in mind until later.)
  • Formation of these hydrogen bonds causes the main chain to adopt regular secondary structures called a helices and b sheets.

alpha helix

• See Nelson & Cox, Lehninger Principles of Biochemistry, Box 6-1 (p. 165) for explanation of right-handed and left-handed helices.
• intrachain hydrogen bonds between the peptide bond elements 4 residues apart in the primary sequence, from the carbonyl C=O of residue n (the hydrogen bond acceptor) to the H-N of residue n+4 (the hydrogen bond donor). (alpha_helix).
  

Figs. 6-4a and 6-4b (Nelson & Cox, Lehninger Principles of Biochemistry): 2 views of the a helix NOTE: Figs. 6-4a and b were corrected in the 4th printing of Lehninger Principles; corrections are in a PDF link ("Errata") on left sidebar on textbook website: http://www.worthpublishers.com/lehninger/.

• Figs. 6-4c and d (Nelson & Cox, Lehninger Principles of Biochemistry): axial view and space-filling model of the a helix (alpha_helix).
  • NOTE: "Core" of alpha helix does NOT have empty space -- atoms in center are in close contact.
  • Where are the side chains (R groups) on the residues in an a helix, relative to the helix axis?

• protein a helices almost always right-handed due to steric problems of L-amino acids in a left-handed helix. (So what handedness of helix would you expect a polymer of D-amino acids to adopt?)
• f and y angles of all residues in a-helical region of a polypeptide are close to the same, f = about -60^o, and y = about -50^o
• n = number of residues per turn of a helix = 3.6 residues / turn (360^o)
  • hydrogen bond goes from carbonyl O at the "end" of one residue to amide NH at the "beginning" of the 4th residue along the chain
• pitch of a helix = distance along helix axis for 1 full turn = 0.54 nm (5.4 angstroms) / turn
• rise of a helix = distance along helix axis per residue = pitch / n = 0.15 nm (1.5 angstroms) / residue
  (It's not that these numbers are so important in themselves, but the comparison later with the same parameters for the collagen helix and the resulting differences in properties of the 2 kinds of helix will be relevant.) 

(left) The peptide bond has a dipole moment. Because all the hydrogen bonds in an a helix are oriented along the helix axis, all the peptide bond units are also aligned in the same orientation along the helix axis. Because the peptide bond has a dipole moment arising from the polarity of the NH and C=O groups (see above), the helix itself has a dipole moment that runs the length of the helix with the amino terminal end of the helix carrying a partial positive charge and the carboxyl terminal a partial negative charge.  Helix axis: (d +)N --> C(d -) (See also Lehninger Principles Fig. 6-6.) The helix dipole moment plays an important role in binding charged ligands to proteins, as we shall see later.

•  Amino acid sequence affects stability of an a helix:
  • electrostatic repulsion/attraction between adjacent residues with charged R groups
  • bulkiness of adjacent R groups
  • interactions between residues spaced 3 or 4 residues apart (same side of helix) (see Lehninger Principles Fig. 6-5)
  • glycine rarely found in a helices (or b conformation) because in less structured surface loops glycine has SO much more conformational freedom than other kinds of amino acid residues that it's entropically just too unfavorable to constrain glycine in regular secondary structures
  • occurrence of Pro residues
    • amide N of Pro has no H for hydrogen bonding, so there's a carbonyl 4 residues earlier with nobody to hydrogen bond to
    • Nevertheless, though it's rare, there ARE occasional Pro residues found in real proteins
      in a-helices.
  • interaction between R groups at ends of helix and the dipole of the helix
    • – charged R groups often near N-terminal (d+) end of helix (increases stability of helical structure)
    • + charged R groups often near C-terminal (d –) end of helix (increases stability of helical structure)
      

 beta conformation

• b conformation more extended conformation than a-helix, with backbone zig-zagging at the (tetrahedral) a carbons
• stabilized by interchain hydrogen bonds, between 2 or more sections of either the same polypeptide backbone (folded back on itself) or different polypeptides backbones
• amide N-H from one section of backbone hydrogen-bonded to carbonyl O from another section of backbone
• no fixed relationship in amino acid sequence between residues hydrogen-bonded to each other, just 2 sections of backbone lined up next to each other
• regular, repetitive pattern, with similar (f,y) coordinates for all the residues
• hydrogen bonds are more or less perpendicular to the strand direction
• strands/sections of backbone hydrogen-bonded to each other can be running "parallel" (N --> C in same direction) or "antiparallel" (N --> C in opposite directions)

• b-conformation of polypeptide chains. In lefthand drawings, a C atoms are red. Righthand drawings are from Fig. 6-7 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.), with N atoms blue, O atoms red, alpha C atoms gray, R groups purple balls. (Note that early printings of the book had arrows for chain direction (N--> C) pointing in the wrong directions!)

  • Note "pleats" that result from the angles at the tetrahedral alpha C atoms in the backbones.
    • Multiple strands in b conformation --> "pleated sheets"
  • Where are the R groups in a b strand? Relative to the "folds/creases" in a pleated sheet, where are the R groups?

antiparallel b-sheet:   Strands are oriented such that N --> C directions are opposite. chime routine: (anti_parallel_beta_sheet)

parallel b-sheet:   Strands are oriented such that N --> C directions are the same. chime routine: (parallel_beta_sheet)

• Compare the hydrogen bonds in the antiparallel vs. parallel b sheets above. Which type of b structure would you predict has the stronger hydrogen bonds?

COMPARISON OF a HELIX AND b-CONFORMATION

•  The structures of most proteins contain combinations of a-helices and b-sheets as well as less regular structures of the backbone.
• Successive secondary structural elements in the primary structure are connected by turns and loops, which permit changes in backbone direction.

Loops

• Loops have irregular lengths and shapes and are on the surface of the protein.
• backbone groups in the loop do not usually form hydrogen bonds to each other, but do form hydrogen bonds to water.

b-turns (also called reverse turns, b-bends, or hairpin turns) 

• abrupt changes in direction of chain, connecting two antiparallel b strands
• result in 180^o turn in direction of polypeptide chain
• involve intrachain hydrogen bonds within a 4-residue section of chain

• If you were folding a protein and wanted to make a sharp bend in a polypeptide backbone, what type(s) of amino acid residue might you choose? Why?

• Glycine very common in b-turns because it is so small (R group presents little steric hindrance in a tight turn).

• Proline also very common in beta-turns
  • has one less "unsatisfied" hydrogen bond donor (imino N in peptide bond has no H)
  • always has the correct phi angle (-60^o), and peptide bonds involving imino N of Pro more readily assume the cis configuration, giving a tight turn
  • The vast majority of peptide bonds in proteins (> 99%) have the trans configuration, but about 6% of peptide bonds involving proline's imino N are in cis configuration; many of these occur in b-turns, where the cis configuration contributes to making an "elbow" (turn) in the backbone.

Fig. 6-8b (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): trans and cis isomers of peptide bond involving imino N of a Pro residue

Fig. 6-9 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): Ramachandran plots for a variety of secondary structures. Part (b) shows (black dots) the actual (f,y) coordinates for all the amino acid residues except Gly residues in the enzyme pyruvate kinase.

 Schematic Conventions in Representing Secondary Structural Elements

This image illustrates the conventions used to represent a-helices (green), b-strands (red) and turns (white) in a ribbon diagram. Note that the direction of the b-strand is indicated by the arrow point. Chime routine (closer look): (convention http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Structure/convention.htm).

Simple Protein Structural Motifs 

• Secondary structural elements are connected to each other to form simple, recognizable motifs.
• Some of the most common motifs (helix-loop-helix, bb connected by a hairpin, and bab) are shown in this Chime routine: (motifs http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Structure/motif.htm).

Tertiary and Quaternary Structures of Proteins

• 3-dimensional structures
• stabilized by noncovalent interactions (and for SOME extracellular proteins, disulfide bonds, which are covalent)
• Globular proteins:
  • water-soluble proteins (unless they're located in biological membranes) with a compact, roughly spherical shape
• Fibrous proteins:
  • proteins whose polypeptide chains are assembled into elongated rope-like (cable-like), or sheet-like structures, usually water INsoluble
  • have repeating motifs (patterns) of a particular 2^o structural element

 Fibrous Proteins

• a-keratins (examples: mammals' hair (including wool), hooves, nails, rhinoceros horns, much of the outer layer of skin)
  • evolved for strength
  • 2^o structure: a helix (right-handed)
  • supersecondary structure: 2 polypeptides with right-handed a helices, coiled together in a left-handed twist to give a supercoil (superhelix, coiled coil); the left-handed twisting around each other slightly changes the number of residues per turn in the individual right-handed a helices, from 3.6 to about 3.5 residues per turn. Result: a "heptad repeat" structure in the individual chains in which the positions of the side chains repeat after 2 turns (diagnostic of a coiled coil). Coiled coils are found in many globular proteins, too -- we'll see them again later in the course.
  • higher levels of assembly of multiple polypeptide chains = quaternary structure
  • Fig. 6-11a & 6-11b (Nelson & Cox, Lehninger Principles of Biochemistry):  Structure of hair

• What part of the structure of 1 polypeptide chain is actually in contact with what part of another polypeptide chain in a coiled coil (2 a helices winding together)?
• a keratins are rich in Ala, Val, Leu, Ile, Met and Phe. What kinds of noncovalent interactions stabilize the supersecondary structure (supercoil) IN a-KERATINS? (Other types of noncovalent interactions can also be involved in stabilizing other proteins' coiled coils.)
• There's also Cys in a keratins (varies with the particular structural role -- hair has less Cys, rhinoceros horns have more Cys).
• What difference in properties is most obvious between hair and rhino horns?
• Besides noncovalent interactions involving burying hydrophobic R groups out of H₂O, what other kinds of bonds/interactions between chains are involved in stabilizing the coiled coils?
• For a-keratins like those found in hair, with less Cys than in "hard" a-keratins, would you characterize the structure as "stretchy", or as "rigid"? How does that relate to how extended the polypeptide chain is in a helices, vs. how extended a polypeptide chain CAN be?

• Collagen
  • ~1/4 of all of the protein in the body (gelatin comes from collagen)
  • structural protein in connective tissues: tendons and sheets that support the skin and internal organs
  • bones and teeth made by adding mineral crystals to collagen
  • Structure: individual polypeptide chains have unique secondary structure: collagen helix
    • left-handed
    • pitch = 1 nm (10 angstroms) per turn
    • n = 3 residues per turn
    • rise = 3.3 angstroms (0.33 nm) / residue
  • Supersecondary structure = 3-stranded coiled coil ("tropocollagen"), a triple helix
  • 3 left-handed collagen helices coil together in a right-handed twist
    • Note theme: strength from the supercoil twist being opposite direction from twist of individual helices
  • Fig. 6-12 (Nelson & Cox, Lehninger Principles of Biochemistry):  Structure of collagen
    (a & b) single chain, in left-handed collagen helix
    (c) tropocollagen, 3 polypeptide chains in 3 different colors
    (d) 3 stranded supercoil looking down the axis, Gly residues in red (see below)

• Why does collagen fold into this unique 2^o and 3^o structure?
• 1^o structure (long polypeptide chains, >1000 residues)
• rich in Pro, hydroxyPro (HyPro or Hyp) and Gly
• conformation of a single collagen helix similar to that of a synthetic polymer of proline residues (polyproline)
• much of amino acid sequence being repeating tripeptide sequences: -Gly-X-Pro- or -Gly-X-HyPro-
• Every third amino acid MUST be glycine -- it's the only residue small enough to fit in the interior of the triple helix.
• much more proline and hydroxyproline than in other proteins --> rigidity (lack of flexibility)
• proline just the right shape for the collagen triple helix (see Chime routine below)
• Hydroxyproline critical for collagen stability (probably involved in intramolecular hydrogen bonds that may involve bridging H₂O molecules)
• HyPro generated by modifying prolines after collagen chain is biosynthesized
• enzyme (prolyl hydroxylase) that hydroxylates Pro requires vitamin C to assist in the addition of oxygen
• Triple helix assembly stabilized by hydrogen bonds between backbone groups on different chains: Gly N-H --- O=C of X on adjacent chain
• What's the clinical consequence of vitamin C deficiency?
• What's the clinical consequence of a point mutation substituting another amino acid for a Gly in the sequence of a collagen chain?
• What would be the problem with relying on gelatin as your primary source of dietary protein?
  (collagen triple helix)

• Compare the a helix and the collagen helix with respect to how extended the chain is in the helix:
  • pitch [distance along the axis per 360^o turn]
  • n [number of residues per turn]
  • rise [distance per residue along helix axis = pitch / n]
• Discuss the effect of the structural differences in the two kinds of helix on the properties of the proteins -- which is more "stretchy" vs. which is more rigid?

• Fibroin
  • produced by insects (e.g. silk moths) and spiders
  • secondary structure: many parts of the chains in antiparallel b conformation
  • 1^o structure rich in Gly and Ala (or sometimes Ser), alternating in the sequence: -Gly-Ala-Gly-Ala-Gly-Ser-
  • supersecondary structure = extended stacked b sheets
  • stacked sheets stabilized by interdigitating R groups between sheets
  • Fig. 6-14 (Nelson & Cox, Lehninger Principles of Biochemistry): Structure of silk
    • all Gly from both sheets in one layer, all Ala (or Ser) from both sheets in other layer
    • consequence: VERY close packing of R groups between sheets

• Chime routine: (silk)
  
• What kinds of noncovalent interactions are mainly responsible for the stability of the stacked sheet structure IN SILK FIBROIN? (Other types of noncovalent interactions can also be involved in stabilizing other proteins' stacked b sheet structures.)
• Extended nature of the polypeptides in b conformation in the silk structure makes the fabric very strong and non-stretchy, but the sheets can slide along each other if the silk fibers are bent, making the fabric quite flexible. (Some kinds of silk, e.g. spider silk, have regions of the polypeptides with a helices and disordered states interspersed with the b sheet regions, and the coil/disordered regions also impart flexibility.)