lec05.docx

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Department
Biological Sciences
Course
BIOC12H3
Professor
Shelley A.Brunt
Semester
Fall

Description
lec05 nuclear magnetic resonance (NMR) spectroscopy 1. Alternative to crystallization 2. Can also be used to analyze structure of a protein 3. Can use proteins in solution a. No need to form crystals b. Place protein in magnetic field c. Atomic nuclei absorb electromagnetic radiation as the magnetic field is varied d. Absorbance is influenced by neighboring atoms i. And thus can study interaction between atoms in close proximity ii. Combine information with the amino acid sequence iii. Useful for small proteins, but not large proteins Bovine ribonuclease A 1. NMR structure a. Shows protein backbone i. Disulfide bridges in yellow are not seen in crystal structure b. Similar to ribbon diagram structures of ribonucleases from cow and human c. Structural similarity often follows functional similarity i. Similar function results in similar structures Structure of the peptide bond 2. Peptide group a. Defined as two atoms involved in peptide bond i. The carbonyl oxygen atom ii. Amide hydrogen atom iii. Two adjacent alpha carbons b. Has a rigid planar structure i. Consequence of resonance interactions that give the peptide bond a 40% double bond character Resonance structure of the peptide bond 3. Two double bond forms (resonance structures) a. Electrons are delocalized around carbonyl (C=O) b. Electrons are delocalized around nitrogen (C=N) i. Rotation around the C-N bond is restricted due to the double bond nature of the hybrid 4. Result of hybrid structure is that the atoms of the peptide bond lie in the same plane Conformation of the peptide group is restricted to one of two possible conformations due to double bond nature of the peptide bond 1. Trans a. Two alpha carbons of adjacent amino acid residues are on opposite sides of the peptide bond i. Opposite corners of the rectangle formed by the planar group ii. Farther apart 2. Cis a. Two alpha carbons of adjacent amino acid residues are on same side i. Closer together 3. The cis and trans conformation arise during protein synthesis a. Once the peptide bond is formed the two forms are not inter-convertible my rotation around the peptide bond i. Cis is less favorable 1. Less stable due to steric interference between the side chains attached to the alpha carbon 2. Nearly all peptide groups are in trans except at bonds involving amide nitrogen of proline a. Steric interference in cis-proline is not significantly greater than the trans ii. Trans is strongly favored 1. Because there is not a large degree of steric hindrance 4. Enzymes (peptidyl prolyl cis/trans isomerases) catalyze the interconversion of cis and trans at proline residues a. This is done by transient destabilization of the resonance hybrid structure of peptide bond i. Allows for rotation to occur Trans and cis proline 1. Energies are similar due to steric clashes 2. The most common cis peptide bonds are x-pro linkages a. Cis and trans isomers both experience steric clashes with the neighboring substitution and are nearly equal energetically b. Therefore the fraction of x-pro peptide bonds in the cis isomer under unstrained conditions ranges from 10-40 % i. The fraction depends slightly on the preceding amino acid, with aromatic residues favoring the cis isomer slightly rigid planar structure due to peptide bond 1. partial double bond character a. due to resonance structures between carbonyl and C-N bond i. limited rotation occurs around carbons and nitrogens 1. due to double bond characteristics a. point is peptide bond is always going to be shorter than nitrogen to alpha carbon i. what does this allow? torsion angles of the polypeptide backbone 1. phi and psi bond angles are important a. where the flexibility of the amino acid chain occurs b. somewhat free rotations around the Ca-N (phi) and the Ca-C (psi) i. through secondary structure 1. both are 180 when polypeptide is extended 2. torsion angles a. also called ramachandran angles b. describe the rotations of the polypeptide backbone around the bonds between i. N-Ca (phi) ii. Ca-C (psi) 3. Ramachandran plot a. View of the distribution of torsion angles of a protein structure b. Provides overview of allowed and disallowed regions of torsion angle values c. Serves as assessment of the quality of protein 3D structures d. Key point i. If we have a way to predict the ramachandran angles for a particular protein 1. We could predict its 3D structure rotation around the N-C aad C -Cabonds around the N-C -C backbone 1. can be restricted between main chain and side chain as well as between carbonyl oxygens's on adjacent amino acid residues a. peptide bond in extended conformation i. 180 degrees b. omega peptide bond angle i. unstable caused by steric interference between carbon oxygens of adjacent residues c. structures of proteins are about whether or not they can handle the steric hindrance the configuration of most a-carbon atoms of amino acids linked in a peptide bond is A) cis B) L-form C) trans D) both a and b are correct E) both b and c are correct allowed conformations of polypeptides are indicated by the ramachandran (conformation) diagrams 2. ramachandrans a. predictions b. what are the sterically allowed phi and psi bonds c. end up with a ramachandran diagram i. not expected to know how to make a ramachandran diagram 1. determine what kind of bond angle occurs in various conformations a. most likely and range of bond angles you would find in ramachandran conformations i. analyse the potential bond angles possible without creating steric hindrance 1. what is the maximum and most likely range of angles in various structures a. e.g. left handed helices are rare 3. how do you read a ramachandran plot? a. Bond angles? 4. sterically allowed values of phi and psi can be determined a. by calculating the distance between the atoms of a tripeptide at all values of phi and psi for the central peptide unit alpha helix 1. can be right handed or left handed a. left handed helices are rare 2. stabilized by intrachain hydrogen bonds a. where the hydrogen bonds go, determines structure of peptide b. each carbonyl oxygen forms a hydrogen bond with the amide hydrogen of the fourth residue further toward the c-terminus of the polypeptide chain 3. look at structure and see amino acid sequence a. where it is hydrogen bonding  should be able to make predicted structure 4. hydrogen bonds are close to parallel along the axis of the helix a. individually weak b. cumulatively strong 5. becomes especially strong when hydrogen bonds are on the interior of the peptide structure a. if it is on the exterior, it will interact with water 6. carbonyl groups point towards the c-terminus a. each peptide group is polar b. all hydrogen bonds point in the same direction c. entire alpha helix is a dipole with a positive n-terminus and a negative c-terminus 7. ideal structure a. 3.6 amino acid residues per turn i. right handed helix the backbone turns in a clockwise direction 8. phi and psi angles of each residue are similar a. gives the helix a regular repeating structure 9. can change the number of residues per turn a. changes conformation towards a ribbon structure i. what does this mean? ideal alpha helix 1. diagram a. n terminal at the bottom b. c terminus at the top 2. pitch and number of amino acids per turn can vary a. ideal is 3.6 per turn i. There are approximately 3.6 amino acids per turn b. within constraints of ramachandran diagram c. what does pitch mean? i. The pitch is the vertical distance from one turn of the alpha helix to the next one above it. essentially all alpha helices in proteins are right handed 1. left handed very rare 2. right handed more common alpha helix 1. structure gives its charge 2. slide 21 hydrogen bonding scheme for an alpha helix 3. the CO group of residue I forms a hydrogen bond with the NH group of residue i+4 a. see a diagram given to you i. know which carbonyl groups bonding with which (amino / hydrogen?) groups 1. slide 22 right handed helix 1. size from 4 to more than 4 residues with average of 12 a. side chains project outward from polypeptide backbone i. therefore stability is affected by side chain makeup 1. bulky side chains such as tyrosine, asparagines are less common a. steric hindrance 2. small uncharged side chains are often found 3. glycine destabilizes due to unconstrained rotation around alpha carbon a. often the alpha helix begins or ends with glycine 4. proline (rigid side chain) least likely component since it disrupts right hand helix a. how? i. bulky ring structure creates steric hindrance 2. certain amino acids don't see in alpha helices a. bulky side chains tend not to be present i. because of steric hindrance 1. tyrosine and asparagine b. glycine destabilizes due to unconstrained rotation around alpha carbon c. least likely component i. proline 1. disrupts nature of the alpha helix 3. small uncharged side chains are often found in alpha helices alpha helix may have hydrophilic amino acids on one face and hydrophobic on the opposite face 1. amphipathic a. amphipathic vs amphoteric i. amphipathic  has hydrophobic and hydrophilic regions ii. amphoteric  act as an acid and base 1. water 2. helical wheel diagram a. left side more hydrophobic than right side (amphipathic) b. alpha helix wheel shows whether or not it is an amphipathic molecule i. normally 3.6 amino acids per turn secondary structure prediction 1. vision of how a protein may look in terms of alpha helix / beta pleated / random coil domains a. Cylinders / H = alpha helix b. Arrows / E = beta pleated sheet c. Lines / C = random coils liver alcohol dehydrogenase 2. predominantly a beta sheet structure 3. key point is its alpha helices 4. active site of enzyme sitting on top (leucine) near external aqueous environment 5. predict that it is a predominantly charged or polar amino acid a. how? i. Slide 26 6. leucine (blue molecules) are buried within the protein (hydrophobic) align multiple related protein sequences and predict 1. collect sequences from various organisms of the same protein to determine a prediction a. using multiple programs to average predictions 2. beta sheets  whether it is parallel or antiparallel depends on how the protein folds leucine zipper 1. two amphipathic alpha helices interacting to produce extended coil a. hydrophilic faces facing outwards b. hydrophobic faces in contact 2. leucine zipper is a common transcription factor a. creates structure b. allows active sites to interact c. GCN4 (transcription factor for DNA) beta structure 1. includes beta strands and beta sheets a. beta strands i. the polypeptide is almost fully extended 1. 0.32 per residue 2. usually 0.15 in alpha helix b. beta sheets i. multiple beta strands 1. 2 to 22 (average 6) 2. arranged side by side 2. rarely do proteins have isolated beta strands a. since the structure itself is not more stable than any other conformation 3. beta sheets are stabilized by hydrogen bonds between carbonyl oxygens and amide hydrogens on adjacent beta strands a. i.e. between neighboring polypeptide chains i. as compared to within one in alpha helices 4. beta strands vs beta sheets a. structure of beta strand i. fully extended structure (compared to alpha helix) ii. take up more space per residue compared to alpha helix b. beta sheets become multiple beta strands i. average 6 within a sheet ii. exception 1. can form beta sheets from the beta strands of separate subunits in the quaternary structure a. verify c. hydrogen bonding within the strand of proteins i. different ways in which hydrogen bonds form result in parallel or antiparallel conformations d. beta strands in the sheet can be parallel or antiparallel i. antiparallel 1. the hydrogen bond forms with the amide hydrogen and carbonyl oxygen of a single residue in the other strand ii. parallel 1. two different residues a. not straight b. less stable than antiparallel sheets c. tend to be hydrophobic on both sides d. buried in the interior of the protein 2. the hydrogen bond forms with the carbonyl and amide groups of two different residues on the adjacent strand e. sometimes called pleated sheets because the planar peptide groups meet at angles like a fold of an accordion f. typical beta sheet contains from two to 15 individual strands i. with the side facing the protein interior being hydrophobic 1. typical beta sheet has sides facing the hydrophobic protein interior g. some proteins are entirely beta sheets i. most have both sheets and helices ii. sometimes do not form beta sheet until individual strands adopt tertiary structure (brought into close proximity) the side chains on a beta strand are alternatively above and below the plane of the strand 1. stabilized by hydrogen bonds between polypeptide strands idealized beta sheet 2. n terminus to c terminus peptide chain
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