Biochemistry: Exam 2- Primary structure- description of all covalent bonds (mainly peptide and disulfide) linking amino acid residues in a polypeptide chaino Most important element is sequence of amino acidso Determines how a protein folds up into 3-D structure and this in turn determines the protein’s function, as well as its cellular location and evolution Proteins with different functions always have different amino acid sequences If primary structure is altered, the protein’s function will be altered- Secondary structure- refers to particularly stable arrangement of amino acid residues giving rise to recurring structural patterns- Tertiary structure- describes all aspects of 3-D folding of a polypeptideo Weak interactions are important in folding into tertiary structure- Quaternary structure- when a protein has 2+ polypeptide subunits - Driving force for protein foldingo The entropy of the solvent molecules drives the folding of proteinso Solvent is ordered in “clathrate cages” around hydrophobic side chains when protein is unfolded (entropically disfavored)o When protein folds, the hydrophobic side chains cluster together to form the “hydrophobic core(s)” (entropically disfavored ordering of main chain and side chain of protein)o Solvent molecules are excluded from the hydrophobic core(s) leading to an increase in entropy of the solvent moleculeso It is this increase in entropy of the solvent molecules that drives protein folding- Protein folding o 1. Random formation of short stretches of secondary structure o 2. Those regions adopting the native-like secondary structure adhere to form clusters of secondary structure. o 3. Formation of the “molten globule”, where regions of secondary structure are clustered in a close state to the native state, but hydrophobic regions may still be exposed to solvent o 4. Molten globule rearranges to give compact tertiary structure- Other factors that stabilize protein structureo Disulfide bondso Hydrogen bond interactions between Mainchain and mainchain atoms Sidechain and mainchain atoms Sidechain and sidechain atomso Ion pairs Between negatively charged sidechains and positively charged sidechainso Metal atoms- Each protein has a distinctive number and sequence of amino acid residues- Polymorphic- is having an amino acid sequence in humans that differs slightly, experiencing ‘flexibility’ and generates no effect on function- Most proteins contain crucial regions that are essential to their function and whose sequence is therefore conserved- Protein labeling & identification:o FDNB to identify amino-terminalo Edman degradation- labels and removes only amino-terminal residue from a peptide, leaving all other peptide bonds in tact Carried out in machine called ‘sequenator’ that mixes reagents in proper proportions, separates the products, identifies them, and records results Length of polypeptide can be accurately sequenced from this procedureo Overall accuracy of amino acid sequencing declines as length of chain growso Large chains must be broken down and segmented into smaller chains first Disulfide bonds interfere with sequencing procedures Locate these bonds by cleaving with trypsin and then resulting peptides are separated by electrophoresis and compared with original set **when genes are available, sequencing DNA first is a faster process- Spatial arrangement of atoms in a protein is referred to as conformationo Possible conformations include any structural state protein can achieve without breaking covalent bondso Conformations existing are marginally stable and have lowest free energy (Nativeproteins)- Stability can be defined as the tendency to maintain a native conformation- A given polypeptide can theoretically assume countless conformations and as a result, theunfolded state of a protein is characterized by high degree of conformational entropyo Tends to maintain unfolded state along with help from hydrogen bonding interactionso **the interactions that counter-act this unfolding desire include disulfide/covalent bonds and weak interactions (hydrogen, hydrophobic & ionic interactions)- Weak interactions dominate as stabilizing force in protein structure; in general, protein with the lowest free energy is one with the maximum amount of weak interactions- Hydrophobic amino acid side chains tend to cluster in a proteins interioro The presence of hydrogen bonding groups without partners in the hydrophobic core of a protein can be so destabilizing that conformations containing these groups are untenable- Hydrogen bonds have important role in guiding protein foldingo Charged amino acid side chains interact with water and salts when protein is unfoldedo Salt bridges counter-act this and provide stabilityo Ionic interactions limit structural flexibility and confer uniqueness to protein structure- Hydrophobic residues are largely buried away in protein interior core- Number of hydrogen bonds and ionic interactions within protein is maximized; thus, reducing the number of hydrogen bonding ionic groups not paired with suitable partner- Peptide Bond:o Alpha carbons of adjacent amino acids are separated by three covalent bonds arranged as: Ca-C-N-Cao Peptide C-N bond cannot rotate freelyo Rotation is permitted about N-Ca bond and Ca-C bondso The rigid peptide bonds limit the range of conformations possible for a chaino Peptide conformation is defined by dihedral angles reflecting rotation about each of the three repeating bonds in the peptide backbone The angle between phi () and psi () is what we measure to describe conformation  involves C-N-Ca-C bonds with rotation around N-Ca bond  involves N-Ca-C-N bonds Both are defined at +/-180 when polypeptide is fully extended and all groups are in the same planeo The dihedral angles increase as the distal/4th atom is rotated clockwiseo From the +/-180 position, dihedral angle increases from -180 to 0 degrees, at which point the 1st and 4th atoms are eclipsed—rotation can continue from 0 to 180 to return to original positiono Phi and psi can have any values between -180-180, but many are prohibited due to steric interference between atoms in polypeptide backbone and amino acid side chains The conformation in which  and  are both 0 is prohibited; merely a reference point for describing angles- Ramachandran plot: o Is a way to visualize backboneEdihedral anglesEψ against φ ofEamino acidEresidues