Biology 2315 Lecture Notes

Chapter 4: 3-D Structure of proteins

Outline There are 4 levels of protein structure Methods for determining protein structure Secondary structure of proteins The a Helix b Structure Other conformations are found in globular proteins Tertiary structure of globular proteins Quaternary structure Protein folding and stability Protein folding is assisted by Chaperones Protein denaturation and renaturation Collagen, a Fibrous Protein Myoglobin, a Globular Protein Hemoglobin, a globular protein possessing quaternary structure Oxygen binding to myoglobin and hemoglobin Oxygen-binding curves of myoglobin and hemoglobin

Proteins are the main players in the life of a cell. Each protein is a unique sequence of amino acid residues, each of which folds into a unique, stable, three dimentional structure that is biologically functional. Proteins are the most structurally and functionally diverse group of macromolecules. Their vaired functions stem from some key features:

• 1. Proteins are linear polymers built of building blocks clalled amino acids.
  • unlimited order of amino acids makes unlimited variety of proteins.
  • the fundamental amino acid alphabet in proteins is several billion years old.
  • each protein is a unique sequence of amio acids residues linked by peptide bonds.
    • peptide bonds are very stable (in absence of an enzyme, peptide bond can last 1000's yrs)
• 2. Proteins contain a wide range of functional groups.
  • hydroxides, thiols, thioesaters, carboxylic acids, amino groups, phosphates, etc...
  • reactivity of these groups accounts for enzyme activity.
• 3. Proteins can interact with one another and with other biological molecules to form complex assemblies. Molecular cooperation leads to molecular synergism ( proteins in a group act synergistically to generate capabilities not afforded by the individual component proteins).
• Conformation = spatial arrangement of atoms that depends on rotation of bonds. Can change without breaking covalent bonds.
• Since each residue has a number of possible conformations, and there are many residues in a protein, the number of possible conformations for a protein is enormous.
• Native conformation = single, stable shape a protein assumes under physiological conditions.
  
• In native conformation, rotation around covalent bonds in polypeptide is constrained by a number of factors ( H-bonding, weak interactions, steric interference).
  
• Biological function of proteins depends completely on its conformation. In biology, shape is everything.
  
• Proteins can be classified as globular or fibrous.
• Some proteins are quite rigid, while other show limited flexibility
  • rigid proteins are often structutral proteins
  • flexibility is useful in making hinges, springs, and levers

  

There are 4 levels of protein structure

• Primary structure
  
• linear sequence of amino acids
• determined by nucleotide sequence of gene
  
• held by covalent forces
  
• primary structure determines overall shape of folded polypeptides (i.e primary structure determines secondary , tertiary, and quaternary structures).
  
• Secondary structure
  
  • regions of regularly repeating conformations of the peptide chain (a helices, b sheets)
    
  • maintained by H-bonds between amide hydrogens and carbonyl oxygens of peptide backbone.
    
• Tertiary structure
  
  • completely folded and compacted polypeptide chain.
    
  • stabilized by interactions of sidechains of non-neighboring amino acid residues (fibrous proteins lack tertiary structure)
    
• Quaternary structure
  
  • association of two or more polypeptide chains into a multisubunit protein.

Secondary structure of protein

• Secondary structure is the hydrogen-bonded arrangement of the backbone of the protein. The two most common secondary structures are the a-helix and b-strands (many strands make sheets)

The a Helix

• most frequently observed secondary structure(Fig 4-2).

  
  

• virtually always found as right-handed
  
• generally have 3.5-3.7 residues per turn:
  
  • pitch (advance per turn) = 0.54 nm
    
  • rise (advance per amino acid residue) = 0.15 nm
    
• each carbonyl oxygen (residue n) of polypeptide backbone H-bonded to backbone amide hydrogen of the fourth residue toward C- terminus (n + 4). Therefore first 3 and last 3 residues lack H-bonding partners within helix)
  
• each hydrogen bond closes a loop of 13 atoms
  
• hydrogen-bonds, which stabilize helix, are nearly parallel to long axis of helix; all carbonyl groups point toward C-terminus.
  
  • cumulative effect of many H-bonds within a helix stabilizes this conformation, especially in hydrophobic regions in the interior of the protein, where there are no water molecules to compete for H-bonds.
    
• side chains of amino acids in a helix point outward from the cylinder of the helix. Side chains affect stability of helix:
  
  • alanine has small , uncharged side chain, so fits well within helix, and is often found in helices of globular proteins.
    
  • glycine, has -H side chain, and destabilizes helices because rotation around a-carbon is so unconstrained.
    
  • proline never found in interior of a-helices. It has rigid cyclic side chain which causes steric interference. Also, lacks amide hydrogen for H-bonding with carbonyl oxygen of other a.a. in helix.
    
• a-helices usually end with structures known as helix stop signal (e.g. capping box).
  
  • side chain of N-terminal residue (n) of helix interacts with backbone amide hydrogen of fourth residue of helix, resulting in a conformation whose f and y angles are not compatible with formation of a helix.
    
    • Serine and threonine usually found at initial positioning of capping box, and glutamate is often the fourth residue.
      
• Globular proteins vary in their a-helical content:
  
  • in myoglobin, 75% of residues found in a-helices
    
  • chymotrypsin has very little
    
  • average for globular proteins is 26%.
    
  • Amphipathic helices = have hydrophilic amino acids on one face of helix cylinder and hydrophobic amino acids on opposite face. Such amphipathic helices often located on surface of globular proteins.

b Structure

• Portions of the polypeptide chain that are almost fully extended ( Fig 4.4-6.)
  
• Includes b strands and b sheets. b sheets consist of multiple b strands arranged in sheets.
  
• Stabilized by H-bonding between carbonyl oxygens and amide hydrogens on adjacent b strands (of same or different polypeptide).
  
• b strands can be parallel or antiparallel. Parallel strands less stable because of distorded H-bonds.
  
• In b sheets, side chains point alternately above and below plane of sheet.
  
• b sheets may contain 2-15 strands, and can form barrel shaped structures.

  
  

• Many globular proteins contain regions of b structure. Rubredoxin composed of 7 b strands arranged in 3 antiparallel b sheets.
  
• As in a helices, side of b sheets facing interior is hydrophobic, and surface side is hydrophilic. In cases where entire sheet is in interior, all amino acids composing sheet are hydrophobic.

Other conformations are found in globular proteins

• Nonrepetitive regions
  
  • sometimes called random coils. but are actually stable, highly ordered structures.
    
  • connect secondary structures; provide directional changes necessary for proper folding (Loops, turns, and hairpin loops) (Fig 4.7).
    
  • most globular proteins have many residues in nonrepeatative conformations (as much as in helices and strands).
    
  • least conserved areas of protein
    
  • hairpin loops in the variable domains of the antibody molecule bind to antigens. High mutation rates observed function to generate antibody diversity. This process, in conjunction with alternative splicing of genes, allows an organism with a finite number of antibody genes to generate an almost unlimited number of antibodies capable of recognizing a very diverse population of antigens.

Motifs

• Supersecondary structures; recognizable combinations of a helices and b strands that appear in a number of different proteins (Fig 4-8 and 4.9).
  
• Sometimes have a particular function
        a. helix-loop-helix
        b. bab unit
        c. hairpin
        d. Greek key

Globular proteins:

• Water soluble; compact, spherical; hydrophobic interior and hydrophilic surface.
  
• Indentations specifically and transiently bind other compounds (active and allosteric sites).
  
• Most enzymes are globular.
• Most proteins are globular. Its the focus of this chapter.

Fibrous proteins:

• Static molecules; provide mechanical support
  
• Typically water insoluble.
  
• Usually built on single repetitive structure assembled into cables or threads
  
  • e.g. a-keratin, collagen, silk.

Tertiary structure of globular proteins

• Result from folding of polypeptide into closely packed, nearly spherical shape.
  
• Stabilized primarily by noncovalent interactions (mostly hydrophobic effect) between side chains of amino acid residues (Fig 4.12).
  
• Lots of variety in tertiary structure.
  
• Many globular proteins composed of independently folded globular units called domains. Domains consist of combinations of motifs (range in size from 30-300 a.a.)
         a. b meander
         b. a/b barrel
  
• Small proteins usually contain one domain. Larger proteins may contain more than one.
  
• Often, each domain has a particular function. In multifunctional enzymes, each catalytic activity associated with different domain.
  
• Regions between domains often form crevices that serve as binding sites (e.g. antibody proteins).

Quaternary structure

• Limited to proteins with multiple subunits.

  

• Each subunit called a monomer. A multisubunit protein is called an oligomer.
  
  • subunits in an oligomeric protein always have defined stoichiometry.
    
• Monomers may be identical or different.
  
• When several metabolic reactions are catalyzed by oligomer, its called a multienzyme complex.
  
• Subunits of oligomer usually held together by noncovalent forces. Usually hydrophobic interactions, but electrostatic forces may also be involved.
  
• A large proportion of globular proteins have quaternary structure. Probable reasons are:
       1. more stable than dissociated subunits.
       2. active sites of some oligomers formed by amino acids on adjacent polypeptides.
       3. likelyhood of errors greater for long polypeptides. Therefore more efficient to synthesize several smaller subunits
       4. 3D-structure changes when proteins bind ligands. Critical step in biological activity of many proteins (i.e regulatory function)

Methods for determining protein structure
Primary structure obtained by protein or DNA sequencing. The 3-D conformatiuon determined by X-ray crystalography and NMR.

• X-ray crystalography
  
  • beam of parallel X-rays aimed at protein crystal. Diffraction of X-rays by electrons is recorded on film or by electronic detector.
    
  • mathematical analysis of diffraction pattern, together with knowledge of primary structure, used to deduce 3-D conformation.
    
  • has followed development of computers
    
  • today, limiting factor is preparation of protein crystals.
    
  • crystals have 20-50% water molecules in them. Can diffuse ligand to show active sites.
    
  • proteins retain native conformation in crystal.
    
• Nuclear magnetic resomance (NMR):
  
  • permits study of proteins in solution
    
  • does not require preparations of crystals
    
  • better suited for small proteins

Protein folding and stability

• As protein folds, first few interactions intitiate subsequent interactions by assisting in the alignment of groups. This process is called cooperative folding (i.e. formation of one part of a structure leads to the formation of the remaining parts of the structure).
  
• Protein folding and stabilization depend on several noncovalent forces, incuding, hydrophobic effect, H-bonding, charge-charge interactions, and van der Waals forces.:
  
• Hydrophobic effect: water molecules interact more strongly with each other than with nonpolar side chains. This leads to side chains being excluded from interaction with water and are forced to aggregate into interior, causing protein to fold.
• Overall increase in entropy is driving force for protein folding. Sections of polar backbone that are forced into interior of protein can be neutralized by H-bonding (allows stable secondary structures to form), or charge-charge interactions.
• Collectively, noncovalent forces provide stability of native conformations. However, the weakness of individual noncovalent interaction gives proteins resilience and flexibility to undergo small conformational changes.
  
• During protein folding, elements of secondary structure probably form first and then fold into tertiary conformation.

Protein folding is assisted by Chaperones

• Proper folding is not random. Its a cooperative, sequential process, in which formation of first few structural elements assists in alignment of subsequent structural features. Folding pattern and final conformation depend on primary structure. For large polypeptides, folding is assisted by molecular chaperones.
  
• Chaperone prevent formation of incorrectly folded intermediates that may trap polypeptide in an aberrant form. Form stable complexes with surfaces on polypeptides chains that are exposed only during synthesis, folding and assembly.
  
• Even in the absence of chaperones, protein folding is spontaneous.
  
• How proteins fold exactly remains a relatively mysterious process.

Protein denaturation and renaturation

• Denaturation = disruption in the native conformation of a protein, with loss of biological activity (Fig 4.18).
  
• Very few H-bonds need to be broken for denaturation to occur.
  
• Denaturation can be caused by heating or changes in pH.
  
• Chaotropic agents and detergents also denature proteins (only disrupt weak interactions). Effects of these chemicals can sometimes be reversed.
  
• High [ ] of chaotropic agents (urea, guanidinium chloride) and detergents denature proteins by disrupting hydrophobic interactions.
  
• Complete denaturation often requires cleavage of disulfide bonds (using mercaptoethanol).
  Fig 4-20: denaturation and renaturation of ribonuclease A.
  
  • demonstrate that correct disulfide bonds form only after proteins fold into native conformation.

Collagen, a Fibrous Protein

• Major component of vertebrate connective tissue (25-35% total protein in mammals)
  
• Diverse in form and function:
  
• collagen in tendons: stiff, rope-like fibers with high tensile strength.
  
• collagen in skin: loosely woven fibers; permits expansion in all directions
  
• Consist of 3 left-handed helical chains coiled around each other to form right-handed supercoil. More extended than a-helix.
  
• Collagen helix stabilized by interchain H-bonds. Has -Gly-X-Y- repeating sequence. Glycine residues in each triplet located along central axis and form H-bonds with X residue of adjacent chain.
  
• No intrachain H-bonds in collagen helix.
  
• Collagen contains modified amino acids: 4-hydroxyproline and hydroxylysine. Modifications are post-translational.
  
  • hydroxylation of proline and lysine catalyzed by enzymes which require ascorbic acid (Vit C). Vit C deficiency results in improper formation of collagen resulting in scurvy (skin lesions, bleeding gums, loose teeth, fragile blood vessels etc...).
    
• Collagen is a glycoprotein: hydroxylysine residues are covalently bonded to carbohydrate residues.
  
• Collagen fibers are rigid and have high tensile strength resulting from covalent cross-links between collagen molecules.

Myoglobin, a Globular Protein

• Functions by selectively and reversibly binding to O₂ in muscles.
  
• Contains heme prosthetic group (Fig 4-17)(source of red colour in muscles and blood).
  Prosthetic group = protein-bound organic molecule that is essential for the activity of protein.
  
  • heme consists of protoporphyrin ring (planar) complexed to iron (Fe²⁺). Iron forms a complex with six ligands: 4 N of protoporphyrin ring, His-93, and O₂.
    
• Myoglobin is member of globin multigene family, which also includes hemoglobins and leghemoglobin from plants.
  
• Sperm-whale myoglobin consists of 8 a-helices connected by short nonrepetitive segments. Heme prosthetic group wedged into hydrophobic cleft (essential for reversible binding to O₂). Accessibility of iron to O₂ dependent on slight movement of nearby amino acid side chains.

Hemoglobin, a globular protein possessing quaternary structure

• Functions within erythrocytes to transport O₂ from gas-exchange organs to other tissues (300 million molecules per rbc).
  
• Oligomeric protein (Fig 4-20): a₂b₂. Both chains are similar and each has heme prosthetic group identical to myoglobin.
  
• Quaternary structure of hemoglobin responsible for oxygen-binding properties not possible for myoglobin.

Oxygen binding to myoglobin and hemoglobin

• Oxygen binds reversibly to heme
  Terminology
  
  • Oxygenation = reversible binding of oxygen
    
  • deoxymyoglobin = oxygen-free myoglobin
    
  • Oxymyoglobin = oxygen-bearing myoglobin
    
• Similar designations for hemoglobin
  
• Hydrophobic heme group buried in hydrophobic pocket but there are two polar residues situated near heme group: His-64 and His-93.
  
  • in oxymyoglobin iron bound to 6 ligands to form octahedron: 4N's, His-93, and O₂, which is bound between iron and His-64(Fig 4-22).
    
  • hydrophobic pocket helps keep heme in place and prevents complete transfer of an electron from Fe²⁺ to O₂, and forces the return of the electron to the iron atom when oxygen dissociates, thereby assuring reversible binding of O₂ for transport.

Oxygen-binding curves of myoglobin and hemoglobin

• Fractional saturation (Y) is plotted against partial pressure of gaseous oxygen (pO₂).
• Y = fraction of total molecules that is oxygenated (Fig 4.24).

  
  

• Oxygen binding curve for myoglobin is hyperbolic, indicative of single ligand binding to macromolecule.
  
• Hemoglobin has sigmoidal oxygen binding curve, indicating that more than one O₂ binds per molecule.
  
  • hemoblobin can bind up to 4 O₂, 1 per heme group.
    
  • shape of curve suggest that binding of first O₂ facilitates binding of O₂ to other hemes. The oxygen affinity of hemoglobin increases as each O₂ is bound (positive cooperative binding).
    
  • cooperative binding related to changes in protein conformation: binding of oxygen to hemoglobin causes movement that disrupts ion pairs within protein and favors conformation with higher oxygen affinity.
    
• P50 is the partial pressure at half saturation: measures affinity of protein for O₂.
  
  • the heme myoglobin has a much higher affinity for O₂ than the heme hemoglobin because of differences in microenvironment
    
    • Differential affinities for O₂ between myoglobin and hemoglobin lead to efficient system for O₂ delivery from lungs to muscles:
      
    • at high pO₂ (as in lungs) both myoglobin and hemoglobin become saturated.
      
    • at low pO₂ (as in capillaries) hemoglobin looses O₂ which is picked up by myoglobin.
• Both H⁺ and CO2, which themselves bind hemoglobin, affect the affinity of hemoglobin to O₂ by altering the 3-D structure.
  
  • Bohr Effect: an increase in [H⁺] reduces O₂ affinity of hemoglobin. Actively metabolizing tissue release H+, promoting O₂ release from hemoglobin (Fig 4.24 and Table 4.1).

  
  

  • Also, in actively respiring tissue, lots of CO₂ is produced. It in turn forms carbonic acid (pka 1 pH unit below blood pH). As a result, 90% of dissolved CO₂ is present as carbonate ion, releasing H⁺. As a result of increase of in [H⁺], hemoglobin looses O₂ in actively respiring tissue. In the lungs, CO₂ exhaled, blood pH increases promoting binding of O₂ to hemoglobin.
    
• Hemoglobin also binds BPG (2,3-bisphosphoglycerate) (Fig 4.25). Binding of BPG to hemoglobin reduces affinity of hemoglobin to O₂. This is important, b/c if BPG not bound to hemoglobin, hemoglobin will not loose O₂ to myoglobin in metabolizing tissues (Fig 4.27). Fetal hemoglobin has less of an affinity for BPG therefore it binds O₂ more strongly, enabling it to pick up O₂ from maternal blood across the placenta (Fig 4.28).

End of chapter questions: 2, 4, 5, 6, 8, 11, 14, 19, 21, 22, 25, 27, 33, 35 40, 43, 44, 50.