2 - Lecture Guide

Review of Lecture 2 Questions to think about…. Are ?-helices found in proteins almost always right-handed or left-handed? MOSTLY RIGHT HANDED To what level of structure do ?-helices belong? SECONDARY, HELD TOGETHER BY HYDROGEN BONDS VIA CARBONYL AND AMIDE WHERE YOU HAVE 3.6 RESIDUES PER TURN, THIS IS EQUIVALENT TO 5.6 ANSTROMS What does it mean to say a protein is oligomeric? MADE UP OF MORE THAN 2 POLYPEPTIDES TO MAKE A WORKING SUBUNIT Why is proline not often found in ?-helices of proteins? BECAUSE IT HAS A HETEROCYCLIC PYROLIDINE RING WHICH INDUCES MAJOR TURNS IN BETA SHEETS. WITH THIS AMINO ACIDS, THERE IS MINIMAL HYDROGEN BONDING AND IF THERE A TURN, ITS SIDE CHAIN IS EXPOSED SO WON’T BE STABLE. Why is glycine not commonly found in ?-helices of proteins? BECAUSE IT HAS A SMALL R GROUP THAT CANNOT FROM HYDROGEN BONDS WITH NEIGHBOURING RESIDURES. Why can’t psi (?) and phi (?) both be zero? AN ANGLE OF ZERO SUGGETSS THAT THE ATOMS HAVE OVERLAPPED. THIS IS IMPOSSIBLE BECAUSE OF STERIC HINDERANCE. What is the difference between a domain and a motif? DOMAIN IS PART OF A PROTEIN SEQUENCE AND STRUCTURE THAT CAN EVOLVE, FUNCTION, AND EXIST INDEPENDENTLY OF THE REST OF THE PROTEIN CHAIN. A motif is a conserved region of DNA sequence prevalent in organisms that code the same protein. The process by which a protein in a random coil conformation assumes its native shape is called RENATURATION. HSP70S AND HSP60S (CHAPERONINS) are the proteins that assist in the cellular process of protein folding such that a large fraction of the protein molecules affected achieve their native conformation and are thus biologically active. A region in a globular protein characterized by negative ? and ? values is a region of ______. A segment of a protein chain in the ?-helical conformation contains 20 residues. How long is this segment? WE KNOW THAT 3.6 RESIDUES EXISTS IN EVERY TURN… ALL YOU DO IS DIVIDE 20 RESIDUES BY 3.6 RESIDUES AND YOU GET ~6 TURNS. NOW ALL YOU DO IS MULTIPLE THIS VALUE BY 5.6 ANGSTROMS TO GET THE ACTUAL LENGTH. How many turns are in this helical segment of 20 amino acids? WE KNOW THAT 3.6 RESIDUE FOR EVERY HELICAL TURN… SO 20 / 3.6 IS AROUND 6 TURNS – 7 IS OVER ESTIMATING The approximate molar mass of an oligomeric protein was found by gel-filtration to be 8 x 104. the native protein was shown to be composed of a single type of protein chain (monomer), the molar mass of which was determined by SDS-PAGE to be 1.9 x 104. Speculate on the oligomeric composition of the native protein. THIS SUGETSS THAT THE OLIGOMERIC CONSISTS OF 4 EQUALLY SIZED SUBUNITS OR POLYPEPTIDES. THE PROTEIN IS HELD TOGETHER BY NONCOVALENT BONDS WHICH WERE DISRUPTED BY THE SODIUM DODECYL SULPHATE DETERGENT, THUS SEPATING INTO FOUR DIFFERENT PARTS. What bonds or interactions are disrupted upon denaturation? Proteins can be denatured using strong acids/bases, PH changes, changing the temperature or applying heat, adding organic solvents such as alcohol or acetone, detergents such as SDS, reducing agents such as beta-mercaptoethanol which disrupts covalent disulfide bonds, chaotropic agents such as urea which disrupts non-covalent bonds by allowing water to solvate hydrophobic regions or regions where vander waal forces and hydrogen bonding are know to occur, and you can denature a protein simply by applying mechanical stress. HYDROPHOBIC, HYDROGEN BONDING, DISULFIDE BRIGDES (MERCAPTOETHANOL AND DIETHRITOL DTT), ELECTROSTATIC FORCES. BASICALLY ANYTHING STABILIZING THE SECONDARY AND TERTIARY STRUCTURE. When denatured proteins refold in the presence of low levels of detergent or denaturant, they form many incorrect disulfide bonds. What does this tell you about the process of protein refolding? Protein folding is a highly specific process involving external sources that assist its proper folding such as chaparonins. LIKEWISE, CORRECT FOLDING MUST OCCUR FROM THE PRIMARY STRUCTURE AND PROCEED ON FORWARD. Which amino acids will usually be found in the interior of a globular protein? Which will be found on the exterior? HYDROPHOBIC, HYDROPHILLIC, RESPECTIVELY. Why is it important to know about protein structure in aqueous solutions? This is the physiological environment where they are found. How could you determine if a protein has quaternary structure? USE 3D ELECTROPHORESIS AND THEN 2D ELECTROPHORESIS WITH SDS AS A DETERGENT – sodium dodecyl sulphate polyacrylamide gel electrophoresis. TYPICALLY, THE MW OF THE NATIVE OLIGOMER IS ESTIMATED BY GEL-FILTRATION CHROMATOGRAPHY MW OF EACH CHAIN IS DETERMINED BY SDS-PAGE…THE RATIO OF THE 2 VALUES PROVIDES THE NUMBER OF CHAINS PER OLIGOMER What contributes to the insolubility of fibrous proteins such as ?-keratin and how does this insolubility contribute to the function of these proteins? FIBROUS PROTEINS ARE QUITE RIGID DUE TO THEIR PHI AND PSI ANGLES THAT MAKE THE AMINO ACID SIDE CHAINS POINT OUTWARD WHICH PREVENTS OUTSIDE SOURCES FROM DISRUPTING THE SECONDARY STRUCUTRE, which is the structure held together by the amino acid polymer backbone via HYDROGEN BONDS. IF the secondary structure is disrupted, the forces that hold the protein together in rigid, fibrous form is lost. How many amino acid residues are typically found in a ?-turn and how is this structure stabilized? A BETA TURN WILL HAVE 5 RESIDUES OR 4 RESIDUES (WHERE GLYCINE IS THE THIRD RESIDUE) ALLOWING FOR ABRUPT 180 DEGREE TURNS. THE STRUCTURE IS STABALIZED BY HYDROPHIC HYDROGEN BONDING FORMING PARELLEL AND ANTIPARELLE (MORE STABLE) INTERACTIONS. RECALL THAT IS TURN IS STABILIZED BY CARBOYNL 1 OF OXYGEN AND AMIDE OF RESIDUE FOUR. 4 amino acids caused abrupt 180 degree turns and every third residue is glycine which is stabilized by hydrogen bonding. What are the limitations on the kinds of amino acids found in ?-structures? AMINO ACIDS FACING THE INSIDE ARE HYDROPHOBIC WHILE THE AMINO ACIDS FACING THE OUTSIDE ARE HYDROPHILIC… THE LATTER PART BEING SIMILAR TO ALPHA HELICES. What makes peptide bonds planar? Their PARTIAL DOUBLE BOND PROPERTIES AND ELECTRON DELOCALIZATION – 2 FACTORS. Are the side chains of amino acids in an ?-helix on the outside or inside of the helix? THE SIDE CHAINS ARE ON THE OUTSIDE WHILE THE OTHER PROTIOSN OF THE PEPTIDE SUCH AS THE ALPHA CARBON AND NITROGEN AR EPON THE INSIDE. This property is what makes a-helices fibrous because their back bone (the inner part) is protected. What information does a Ramachandran Plot provide? PHI AND PSI ANGLES OF KNOWN PROTEINS/ Know the interactions that maintain tertiary structure. Electrostatic, covalent, hydrophobic interactions, van der wals forces, disulfide bridhges Be familiar with the different classifications of proteins (functional (transport, enzymatic, defence, support) vs. structural (globular, fibrous) vs cellular localization). Functional: Defence, enzymatic, support, transport. Structural: Globular versus fibrous cellular localization: membrane and soluble Cis and Trans conformations of peptide group: which is the most favourable? Trans is more favourable because the atoms are more spread apart where as cis induces steric hinderance. Know what a ‘homolog’ is. What is a motif? Why is it important to know which amino acids are invariant, conserved or not conserved within protein structure? Know the levels of protein structure. Primary, secondary, tertiary, quaternary . Alpha-helix: know the number of amino acids per turn; the pitch; the rise. 3.6 amino acids per turn which is equivalrent to 5.4 angstroms. What amino acids are incompatible with the alpha helix? – PROLINE Be able to recognize left-handed and right-handed alpha-helices. Beta-sheet: antiparallel vs parallel which is more stable? Know the difference between heteromers and homomers; dimeric versus monomeric. ?? For extra review do the following questions from your text: #9, 10, 11, page 180 #30, 41, page 181