3-D Structure of proteins

OutlineThere 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 amino 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.

Levels of Protein Structure

Interactions in FoldingProtein with itself : Hydrogen bonds Electrostatic interactions Van der Waals interactionsProtein-Solvent: induces burial of hydrophobic residues Hydrophobic residue Hydrophilic residue

Secondary structure of proteinsSecondary 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.

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.

Structureportions of the polypeptide chain that are almost fully extended 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) strands can be parallel or antiparallel. Parallel strands less stable because of distorded H-bonds. in sheets, side chains point alternately above and below plane of sheet. 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 nonrepetative 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. 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. meander 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).

The forces that give rise to the tertiary structure of a protein areIonic bonding hydrogen bonding hydrophobic interaction disulfide bonds

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)

Denaturation The natural or native structures of proteins may be altered, and their biological activity changed or destroyed by treatment that does not disrupt the primary structure. This denaturation is often done deliberately in the course of separating and purifying proteins. For example, many soluble globular proteins precipitate if the pH of the solution is set at the pI of the protein. Also, addition of trichloroacetic acid or the bis-amide urea (NH2CONH2) is commonly used to effect protein precipitation. Following denaturation, some proteins will return to their native structures under proper conditions; but extreme conditions, such as strong heating, usually cause irreversible change. Some treatments known to denature proteins are listed in the following table.

Denaturing ActionMechanism of OperationHeat hydrogen bonds are broken by increased translational and vibrational energy. (coagulation of egg white albumin on frying.)Ultraviolet Radiation Similar to heat (sunburn)Strong Acids or Bases salt formation; disruption of hydrogen bonds. (skin blisters and burns, protein precipitation.)Urea Solution competition for hydrogen bonds. (precipitation of soluble proteins.)Some Organic Solvents (e.g. ethanol & acetone) change in dielectric constant and hydration of ionic groups. (disinfectant action and precipitation of protein.)Agitation shearing of hydrogen bonds. (beating egg white albumin into a meringue.)