Chapter 4 - Protein structure.pptx
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CHAPTER 4: PROTEIN STRUCTURE Week4 Outline Primary structure Secondary structure a-helices b-sheets Tertiary structure Forces Structural determination Quaternary structure Protein folding Levels of Protein Structure https://www.youtube.com/watch?v=1peFJ_-N7V8 Protein structure (primary) Primary (1°)...
CHAPTER 4: PROTEIN STRUCTURE Week4 Outline Primary structure Secondary structure a-helices b-sheets Tertiary structure Forces Structural determination Quaternary structure Protein folding Levels of Protein Structure https://www.youtube.com/watch?v=1peFJ_-N7V8 Protein structure (primary) Primary (1°) structure 1° structure: the sequence of amino acids in a polypeptide chain, read from the Nterminal end to the C-terminal end Refers to the linear amino acid sequence L-R from N- to C-terminal end, e.g. V L R F I V L … Val Leu Arg Phe Ile Val Leu Protein structure (secondary) Protein structure (secondary) Local conformations, maintained by extensive Hbonding that involves components of the peptide bond (carbonyl O and amide H of the peptide back-bone) 2 major kinds of 2o structures found in proteins a-helix – helical, coiled b-sheets – extended “flat” sheets Secondary (2˚) structure 2˚ of proteins: α-helices, β-strands, and turns which are formed by a regular pattern of hydrogen bonds between the amide N-H and C=O groups of amino acids that are near each other in the primary sequence. Two bonds have free rotation: 1) Bond between -carbon and amino nitrogen in residue 2) Bond between the -carbon and carboxyl carbon of residue α-helices, β-sheets and β-turns image credit: OpenStax Biology. -Helix α helix: C=O of one amino acid is hydrogen bonded to N-H of an amino acid that is four down the chain. H-bods are parallel to axis. E.g., C=O of amino acid #1 forms a hydrogen bond to N-H of amino acid #5 Pattern of H-bonds pulls the polypeptide chain into a helical structure Each turn of the helix containing 3.6 amino acids Turn repeat distance is 5.4Å (1 Å = 0.1 nm) The R groups of the amino acids stick outward from the α helix Image credit: Tudor Oprea, UNM (Biomed505) -Helix (Cont’d) Several factors can disrupt an -helix proline creates a bend because of (1) the restricted rotation due to its cyclic structure and (2) its -amino group has no N-H for hydrogen bonding strong electrostatic repulsion caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Asp steric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr Proline – alpha helix breaker and causes ‘kink’ or ‘bend’ -Pleated Sheet Polypeptide chains lie adjacent to one another into a sheet-like structure; may be parallel or antiparallel R groups alternate, first above and then below plane hydrogen bonds form between the backbone of sheets R groups extend above and below the plane The b-sheet Side chains project above and below the plane of the sheet anti-parallel b-sheet Structures of β-reverse turns Glycine found in reverse turns: spatial (steric) reasons Polypeptide changes direction Proline also encountered in reverse turns Secondary (2°) structure Super-secondary structures E.g. bab, aa, b-meander, Greek key (right) b-barrels (below) Some proteins can have multiple domains… Two ways to represent protein structures Space filling model Ribbon structure Protein structure (tertiary) Tertiary (3°) structure Describes the completely folded and compacted polypeptide chain Stabilized by interactions of amino acid side chains in nonneighboring regions of the polypeptide chain. Global 3D peptide structure, with many different types, such as: Globular – often approximated as spheres, tend to be water soluble, polar R on exterior and non-polar inside, often combine a-helix & b-sheets. Fibrous: sheets or fibers, often strong, insoluble, with structural roles Globular Proteins Globular proteins: proteins which are folded to a more or less spherical shape they tend to be soluble in water and salt solutions most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and iondipole interactions most of their nonpolar side chains are buried inside nearly all have substantial sections of -helix and -sheet Fibrous Proteins Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis. They consist of long fibers or large sheets tend to be mechanically strong are insoluble in water and dilute salt solutions play important structural roles in nature Examples are keratin of hair and wool collagen of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels Protein folding Tertiary (3°) structure forces Non-covalent: H-bond, electrostatic interaction, hydrophobic interaction Covalent: disulfide bond Tertiary (3°) structure forces Non-covalent: H-bond, electrostatic interaction, hydrophobic interaction Covalent: disulfide bond Hydrogen bonding in 3o structure Proper protein folding leads to proper binding site & activity 30 Protein structure (quaternary) Quaternary (4°) structure The association of polypeptide chains Several peptide chains (sub-units) combine together E.g. hemoglobin has 4 sub-units labeled a and b (don’t confuse with 2°structure) Hemoglobin vs Myoglobin Myoglobin: in muscle and tissue, no quaternary structure, binds 1 O2 molecule Hemoglobin: in blood, 4 subunits, binds 4 O2 molecules, exhibits cooperativity, and [CO2] and pH affect O2 binding – pKa of His146 decreases when O2 binds (Bohr effect) Myoglobin vs Hemoglobin A comparison of the oxygen binding behavior of myoglobin and hemoglobin. The oxygen-binding curve of myoglobin is hyperbolic, whereas that of hemoglobin is sigmoidal. Myoglobin is 50% saturated with oxygen at 1 torr partial pressure; hemoglobin does not reach 50% saturation until the partial pressure The Bohr Effect The general features of the Bohr effect. In actively metabolizing tissue, hemoglobin releases oxygen and binds both CO and H+. In the lungs, hemoglobin releases both CO and H+ and binds oxygen. 2 2 The Bohr Effect The Bohr Effect 2,3BPG is an allosteric inhibitor of hemoglobin Raises the P50 for deoxy-Hb from 12 to 26 torr Present in RBCs at 1:1 molar ratio with Hb 2,3BPG heme 2,3-BPG effect on hemoglobin A comparison of the oxygen binding properties of hemoglobin in the presence and absence of BPG. Note that the presence of the BPG markedly decreases the affinity of Structure Determination X-ray diffraction of protein crystals Multi-dimensional nuclear magnetic resonance of proteins in solutions and (sometimes) in membranes Increasingly – cryo-electron microscopy, with complex computational processing algorithms Cryo-EM image credit: arXiv:1504.03573 [cs.CV] Structure Prediction De novo or homology-based predictions? Not a solved problem; some sequences are more tractable than others Competitive research: bioinformatics Structure Prediction Very good, but still a lot needs to be done… Structure Prediction An artificial intelligence (AI) network developed by Google AI offshoot DeepMind has made a gargantuan leap in solving one of biology’s grandest challenges — determining a protein’s 3D shape from its amino-acid sequence. DeepMind’s program, called AlphaFold, outperformed around 100 other teams in a biennial proteinstructure prediction challenge called CASP, short for Critical Assessment of Structure Prediction. The results were announced on 30 November, at the start of the conference — held virtually this year — that takes stock of the exercise. “This is a big deal,” says John Moult, a computational biologist at the University of Maryland in College Park, who co-founded CASP in 1994 to improve computational methods for accurately predicting protein structures. “In some sense the problem is solved.” https://www.nature.com/articles/d41586-020-03348-4 Denaturation of proteins Denaturation: the loss of the structural order (2°, 3°, 4°, or a combination of these) that gives a protein its biological activity; that is, the loss of biological activity Denaturation Denaturation (protein unfolding) is caused by: pH changes - side chain electrostatics Mercaptoethanol – reduce S-S bonds Detergent - hydrophobic interactions Heat causes disruptive vibrations Urea/guanidine – H-bonds Denaturation may or may not be reversible Errors in folding can contribute to diseases such as Alzheimer’s and Diabetes type 2 Denaturation Proteins can renature back to the Native structure (if the denaturation was not TOO severe!) – examples “b” Protein Folding Hydrophobic Effect Non-polar species in water reduce entropy as water is forced to organize around them Globular proteins have hydrophobic residues inside Membrane proteins have hydrophobic residues in membrane interior Partly-folded protein - aggregation Protein aggregation linked to numerous neurodegenerative diseases! Protein Folding Chaperones Molecular chaperones: part of a quality control system that aims to ensure proper protein folding OR restore proteins that have become misfolded after various forms of stress (e.g. exposure to heat) Heat-induced denaturation of proteins exposes the nonpolar segments that are normally buried within the protein to aqueous solvent. Chaperones use a variety of strategies to guide the misfolded regions back into place. Image credit: http://physicallensonthecell.org/molecularmachinery/chaperone-aided-protein-folding Molten globule – a third phase of proteins “The term molten globule (MG) was first coined by A. Wada and M Ohgushi in 1983. It was first found in cytochrome c, which conserves a native-like secondary structure content but without the tightly packed protein interior, under low pH and high salt concentration.” http://en.wikipedia.org/wiki/Molten_globule 51 Intrinsically unstructured proteins and their functions H. Jane Dyson & Peter E. Wright Abstract Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold… Nature Reviews Molecular Cell Biology 6, 197-208 (March 2005) | doi:10.1038/nrm1589 Dr. Salman Ashraf 52
