BCHS 3304 General Biochemistry I Midterm 2 Review PDF

BCHS 3304 General Biochemistry I section 11201 Dinler A. Antunes, DSc Assistant Professor of Computational Biology Center for Nuclear Receptors and Cell Signaling Department of Biology and Biochemistry University of Houston Houston - TX Midterm 2 (10/...

BCHS 3304 General Biochemistry I section 11201 Dinler A. Antunes, DSc Assistant Professor of Computational Biology Center for Nuclear Receptors and Cell Signaling Department of Biology and Biochemistry University of Houston Houston - TX Midterm 2 (10/9) will cover all the material from Book chapters 6 and 7 – all sections. Will cover all the material from Secondary Structure up to Myoglobin and Hemoglobin. 2 Review for midterm II Loading…  4 Main Levels of Protein Structure 1. Primary structure 1 = Amino acid sequence, linear order of AA’s. 2. Secondary structure 2 = Local spatial alignment of amino acid backbone without regard to side chains, a-helix, b-strands/sheets, random coil, and b-turns 3. Tertiary Structure 3 = the 3-dimensional structure of an entire polypeptide, fold, biological function and catalytic mechanism. 4. Quaternary Structure 4 = the manner in which the tertiary structures of two or more polypeptide chains of a protein interact, spatial arrangements of subunits (folded chains).  Additional levels of structural organization Primary structure Secondary structure Supersecondary structural motifs Protein domains Tertiary structure Loading… Quaternary structure o wil  Rotation or dihedral angles C -N phi I C -C psi - When viewed down the Ca-N axis, & rotation to the right or clock wise increases the angle of rotation. Must start with the fully extended form which is defined as 180o or -180o  Ramachandran Diagram If you plot on the Y-axis and f on the X-axis, you will plot all possible combinations of f,. 1 sucture? You must know the identities of the different regions of the - favorable or - meaning of islands /white unfuvorable? space - identify 20 structures - Pro + Gly on the outside T rigid flexible  favorable : conform Wo or little steric hind. energ ·. unfavorable : energetically with conformations hinderance steric · too many amino acids (too little white space would make incorrect graph  The Nm Helix Nomenclature C O N = the number of repeating units per turn - - - M = the number of atoms that complete the h-bond cycle O · Example: α-helix h-bonds cycle (n+4th) C=O of AA#1 binds to N-H of AA#5 2 - AA AA #1 #1 AA #2 AA AA #3 #3 AA AA #4 #4 AA AA #5 #5 AA AA #6 #6 ⑳ What is the designation for an a-helix? 3.61 T 3  Different types of Helixes 4.11 6 3.61 31  - b-strand straight b-sheet O b-strand - b-strand O - wonky b-sheet b-strand  b-Sheet Facts Repeat distance is 7.0 Å R group on the amino acids alternate up-down-up above and below the plane of the sheet 2 - 15 amino acids residues long Loading… 2 - 22 strands per sheet Avg. of 6 strands with a width of 25 Å Parallel less stable than antiparallel Antiparallel needs a hairpin turn Tandem parallel needs crossover connection which is right handed sense  Sidechain Locations in Proteins Non-polar sidechains (Val, Leu, Ile, Met, and Phe) occur mostly in the interior of a protein keeping them out of the water (hydrophobic effect) Charged polar residues (Arg, His, Lys, Asp, and Glu) are normally located on the surface of the protein in contact with water. Uncharged polar residues (Ser, Thr, Asn, Gln, and Tyr) are usually on the protein surface but also occur in the interior of the protein. Usually involved in hydrogen bonds with neighbors.  Hydropathy scale Hydropathy scale allows an M o assessment of which amino r acid residues would point e H towards the interior (high y d numbers ) or towards the r o surface (low numbers). p Hydrophobic effects also Mh oo direct transmembrane proteins rb eic where the most hydrophobic H residues are found in y d membrane spanning regions r o 13 p  Protein Structure Determination Experimentally: X-ray crystallography Nuclear Magnetic Resonance (NMR) Cryogenic electron microscopy (cryo-EM) Different basis, protocols, advantages/disadvantages Main objective for all these methods: to determine the 3D structure of a protein! (to determine the atom-level spatial arrangement for all amino acids in the protein) 14  X-ray Crystallography X-rays are bounced off of the protein and deflected by electrons in the various atoms/bonds. Not all atoms deflect the X-ray in the same way (variable intensities on the electron density) The diffraction pattern of the X-rays is measured and an electron density map is created (blue in the figure to the left). Amino acids structures are fit into the electron density. High/“good” ↓ largestest reso. smallestso resolution  X-ray Crystallography advantages Crystalline proteins assume conformations that are very similar to that of the protein in solution (near-native structure) keep same folded structure - - Most independent X-ray crystallography experiments describe the same conformation for the same structure (consistency/reproducibility) - Many enzymes are catalytic active in the crystalline state. Since activity is highly dependent on structure, this is strong evidence that the crystalline conformations must indeed be near-native. - X-ray Crystallography limitations Need for a protein crystal (not always possible, very specific conditions - good are required for each different protein/complex to crystalize) hard - to make crystals There are types of molecules that are harder to crystalize or to analyze (i.e., transmembrane proteins, carbohydrates, IDPs, etc). Resolution limit of obtained crystals (additional data helps!) Protein structures are determined in a “static” state (conformation) "frozen" - - Parts of protein structure might be distorted by crystal packing effects  position ofatoms NMR Spectroscopy - in relation to others Paramagnetic nuclei have interactions with external magnetic field (1H, 2H, 13C, 15N and 31P). Nuclei can absorb energy at particular frequencies (resonance frequencies). Resonance frequencies are sensitive to chemical environment and nearby models that fitdata nuclei. -generating Correlation spectroscopy (COSY) provides interatomic distances between Ensemble of Structures protons that are covalently connected through one or two atoms. Nuclear Overhauser spectroscopy (NOESY) provides interatomic distances for protons that are close in space, but not necessarily connected  NMR spectroscopy advantages No need for crystallization - Can be used to determine the protein structure in solution amanforms - Provides not a single conformation, but an ensemble of conformations , -- Can probe motions over time scales spanning 10 orders of magnitude Resolution can be comparable with X-ray crystallography (NMR spectroscopy results are usually consistent with crystallographic data) NMR spectroscopy limitations Protein size limited to ~100 kD (maybe more in the future…) Raw data can be very difficult to interpret, depending on protein size and flexibility Higher computational cost for model building (fit structure to data) Requires relatively large amounts of pure samples (on the order of several mg) to achieve a reasonable signal to noise level. Cryogenic Electron-Microscopy (Cryo-EM) Different from regular EM in which the samples are dry (not useful for proteins!) Involves fast freezing to extremely low temperatures (-180 to -269 ℃) Proteins are frozen in their native state in solution EM is used to make images of this frozen medium, enabling identification of several protein conformations High resolution structure determination is possible, but requires advanced algorithms data for image processing, clustering and model fitting. - no size limit - Captive wider range of motions 19  Cryo-EM advantages No need for a crystal No need for large amounts of sample (about 0.1 mg) No limit of size or weight (bigger is easier than smaller) Protein conformations in native state Can capture very different conformational states (if they exist in solution) Cryo-EM limitations Molecules are detected in unknown orientations resolution Raw data can be very noisy and difficult to analyze low- Different “shapes” might reflect the same conformation (diff. orientation) Similar “shapes” might reflect different conformations Resolution was inferior to both NMR and X-Ray crystallography Image processing and machine learning methods can be used to cluster conformations, improving model fitting and improving the resolution of predicted structures!! Fast improvement in methodos!!  Protein Structure Prediction Computationally : Protein structure via bonds whais the basis? biochem Ab Initio (from sequence to structure) Fold recognition (Threading) Homology modeling (same function = same folding) - protein conserved as much as Sequence - keeping same fork = same structure 21  Protein structures are stabilized by several different forces Electrostatics, hydrogen bonds and van der Waals forces hold a protein together (tertiary and quaternary structs.). Peptide chains can be cross-linked by disulfides, salt-bridge networks, metal ions, prosthetic groups, or other ligand compounds. Proteins refold very rapidly and generally in only one stable conformation. Hydrophobic effects force global protein conformation and has the greatest effect on structure and stability. 22 Different conditions can cause protein denaturation Heating disrupts protein structure thermal vibrations and disrupt weak bonding forces. (Note that there are heat stable proteins, which have a few more hydrogen bonds and salt bridges – - - networks of “weak” interactions!) - - pH changes lead to denaturation. Protonation of amino acids leads to loss of charge and h-bonding. Detergents associate with the nonpolor amino acids blocking water interactions. Chaotropic agents (e.g., Urea and Guanidinium ions) disrupt hydrophobic interactions by increasing the solubility of nonpolar groups (most commonly used protein denaturants, but mechanism of action not well understood). 23  Proteins fold in a hierarchical way - hydrophic effect is most important during interactions of Proteins can (usually) fold O arrangements Secondarybackbone spontaneously into their native states structure via directed pathways rather than formation random conformational searches (