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Biological Macromolecules Structure and Function of Carbohydrates, Nucleic Acids, Proteins, Lipids Cells as chemical reactors Living organisms obey the laws of chemistry and physics Can think of cells as complex chemical reactors in which many different chemical reactions are proceeding at the same time All cells more similar then different if looked at on the inside! Strip away the exterior and we see that all cells need to accomplish similar tasks and in a broad sense they use the same mechanisms (chemical reactions) Reflects a singular origin of all extant living things! Similarities among all types of cells All cells use nucleic acids (DNA) to store information All cells use proteins as catalysts (enzymes) for chemical reactions A few examples of RNA based enzymes, which may reflect primordial use of RNA All cells use lipids for membrane components RNA viruses, but not true cells (incapable of autonomous replication) Different types of lipids in different types of cells All cells use carbohydrates for cell walls (if present), recognition, and energy generation All cells use nucleic acids (RNA) to access stored information LUCA (Last Universal Common Ancestor) Macromolecules Biotechnology often concerned with the manipulation of cells through the manipulation of the macromolecules contained within those cells DNA Proteins Lipids & Carbohydrates (indirectly) Biologically important macromolecules are “polymers” of smaller subunits Created through condensation reactions Macromolecule Carbohydrates Lipids Proteins Nucleic acids Subunit : : : : simple sugars CH2 units amino acids nucleotides Where do the subunits come from? All cells need a source of the atomic components of the subunits (C, O, H, N, P, and a few other trace elements ) Some cells can synthesize all of the subunits given these atomic components and an energy source Some cells can obtain these subunits from external sources Some cells can convert other compounds into these subunits We will discuss further in section on metabolism and cell growth Carbohydrates All have general formula CnH2nOn (hydrates (H2O) of carbon) A variety of functions in the cell Large cross-linked carbohydrates make up the rigid cell wall of plants, bacteria, and insects In animal cells carbohydrates on the exterior surface of the cell serve a recognition and identification function A central function is energy storage and energy production ! Carbohydrates Cell structure: Cellulose, LPS, chitin Chitin in exoskeleton Cellulose in plant cell walls Lipopolysaccharides (LPS) in bacterial cell wall Carbohydrate Structure Monosaccharides may also form part of other biologically important molecules Carbohydrate Structure Complex carbohydrates built from simple sugars Most often five (pentose) or six (hexose) carbon sugars Numerous –OH (hydroxy) groups can form many types of “cross links” Can result in very complex and highl;y cross linked structures ( cellulose, chitin, starch, etc.) Carbohydrate Structure A Few Examples Triose (3 carbon) Glyceraldehyde Pentose (5 carbon) Ribose Carbohydrate Structure Example of two hexoses Glucose What’s the difference? Both are C6H12O6 Galactose They are isomers of one another! Same formula, but different structure (3D-shape). Carbohydrate Structure Monosacharides can be joined to one another to form disaccharides, trisaccharides, ……..polysaccharides Saccharide is a term derived from the Latin for sugar (origin = "sweet sand") Carbohydrates classified according to the number of saccharide units they contain. A monosaccharide contains a single carbohydrate, over 200 different monosaccharides are known. A disaccharide gives two carbohydrate units on hydrolysis. An oligosaccharide gives a "few" carbohydrate units on hydrolysis, usually 3 to 10. A polysaccharide gives many carbohydrates on hydrolysis, examples are starch and cellulose. Carbohydrate Structure Pentoses and hexoses are capable of forming ring (cyclic) structures. An equilibrium exists between the ring and open form. Linear form Ring (cyclic) form Carbohydrate Structure Monosaccharides can link to form disaccharides Glucose + Fructose Sucrose Carbohydrate Structure Glycosidic Bond β-D Glucose Sucrose (Glucose + Fructose) What does the β mean? Carbohydrate Structure They are isomers of one another α-isomer β-isomer Common small carbohydrates Glyceraldehyde Fructose Ribose Lactose Complex Carbohydrates Cellulose Most abundant carbohydrate on the planet! Component of plant cell walls Indigestible by animals β 1-4 bonds Starch Energy storage molecule in plants Can be digested by animals α 1-6 bonds Cellulose Cellulose is a linear polysaccharide in which some 1500 glucose rings link together. It is the chief constituent of cell walls in plants. Human digestion cannot break down cellulose for use as a food, animals such as cattle and termites rely on the energy content of cellulose. They have protozoa and bacteria with the necessary enzymes in their digestive systems. Only animals capable of breaking down cellulose are tunicates. Starches Starches are carbohydrates in which 300 to 1000 glucose units join together. It is a polysaccharide used to store energy for later use. Starch forms in grains with an insoluble outer layer which remain in the cell where it is formed until the energy is needed. Then it can be broken down into soluble glucose units. Starches are smaller than cellulose units, and can be more readily used for energy. In animals, the equivalent of starch is glycogen, which can be stored in the muscles or in the liver for later use. α-1,6 bonds Complex Carbohydrates Glycogen Branched chain polymer of glucose Animal energy reserve Found primarily in liver and muscle α 1-4 & α 1-6 bonds Glycogen polysaccharides can be linked to other molecules to form glyco-proteins and glyco-lipids Glycoproteins Some examples Polysaccharide component of antibodies has major effect on antibody function Polysaccharides attached to proteins on surface of red blood cells (RBC) determine blood type (A,B,O) Polysaccharides are attached to proteins in the Golgi apparatus through a process of post-translational modification Different types of cells do different post-tranlational modifications More about this later Glycosylation of mAb MW ~150 K Fab express antigen binding regions Fc region interacts with ligands and determines effect. N-linked glycosylation (Asn 297) required for effector function Glycoform structure (N-linked) Consits of a core structure Branched hepatosaccharide GlcNac GlcNac α (1-6) Man GlcNac Man α (1-3) Man GlcNac Glycoform Structure Core required for effector function GlcNAc Fuc GlcNac α (1-6) Man Man α (1-3) Man GlcNAc “outer arm” sugar residues Influence effector functions (ADCC, α-inflammatory, etc) GlcNAc GlcNAc Gal Gal Neu5Ac Neu5Ac Glycolipids Polysaccharides can also be attached to lipid molecules •An outer-membrane constituent of gram negative bacteria, LPS, which includes O-antigen, a core polysaccharide and a Lipid A, coats the cell surface and works to exclude large hydrophobic compounds such as bile salts and antibiotics from invading the cell. O-antigen are long hydrophilic carbohydrate chains (up to 50 sugars long) that extend out from the outer membrane while Lipid A (and fatty acids) anchors the LPS to the outer membrane. Glycolipids Polysaccharides (blue) are also used in animal cells to link surface proteins and lipid anchors to the membrane. Lipids Lipids Fatty acids (Polymers of CH2 units) Glycerol Triglycerides Other subunits (phosphate, choline, etc) may be attached to yield “phospholipids” Charged phosphate groups will create a polar molecule with a hydrophobic (nonpolar) end and a hydrophillic (polar) end Lipids Phospholipids Lipids Function Energy Storage Triglycerides Cell membranes and cell compartments Bi-layer structure Outer or plasma membrane Nuclear membrane Internal structures Er, Golgi, Vesicles, etc. Phospholipid bilayer Hydrophillic heads Hydrophobic tails Steroids Proteins Proteins serve many essential roles in the cell Polymers of amino acids 20 naturally occurring amino acids A few modified amino acids are used The large number of amino acids allows huge diversity in amino acid sequence N = # of amino acids in a protein N20 = # of possible combinations Protein Function Some examples Structure- form structural components of the cell including: Cytoskeleton / nuclear matrix / tissue matrix Movement - Coordinate internal and external movement of cells, organells, tissues, and molecules. Muscle contraction, chromosome separation, flagella……… Micro-tubueles, actin, myosin Transport-regulate transport of molecules into and out of the cell / nucleus / organelles. Lamins, collagen, keratin……. Channels, receptors, dynin, kinesin Communication-serve as communication molecules between different organelles, cells, tissues, organs, organisms. Hormones Protein Function Some examples Chemical Catalyst – serves to make possible all of the chemical reactions that occur within the cell. Defense-recognize self and non-self, able to destroy foreign entities (bacteria, viruses, tissues). Enzymes (thousands of different enzymes) Antibodies, cellular immune factors Regulatory-regulates cell proliferation, cell growth, gene expression, and many other aspects of cell and organism life cycle. Checkpoint proteins, cyclins, transcription factors Protein Structure Polymers of 20 amino acids All amino acids have a Common “core” Amino end (N end) Acid end (C end, carboxy end) Linked by peptide bond 20 different side chains Properties of amino acids amino acids: acidic basic hydrophobic Amino acids all have The same basic structure Chemical properties of the amino acids yield properties of the protein! Properties of amino acids Protein Structure The 3-D shape and properties of the protein determine its function. Shape and properties of protein determined by interactions between individual amino acid components. Four “levels” of protein structure Primary (Io), secondary (IIo), tertiary (IIIo), and quaternary (IVo) (sometimes). Levels of Protein Structure I0 (primary) structure Linear order of amino acids in a protein: 1AASXDXSLVEVHXXVFIVPPXILQAVVSIA 31 T T R X D D X D S A A A S I P M V P G W V L K Q V X G S Q A 61 G S F L A I V M G G G D L E V I L I X L A G Y Q E S S I X A 91 S R S L A A S M X T T A I P S D L W G N X A X S N A A F S S 121 X E F S S X A G S V P L G F T F X E A G A K E X V I K G Q I 151 T X Q A X A F S L A X L X K L I S A M X N A X F P A G D X X 181 X X V A D I X D S H G I L X X V N Y T D A X I K M G I I F G 211 S G V N A A Y W C D S T X I A D A A D A G X X G G A G X M X 241 V C C X Q D S F R K A F P S L P Q I X Y X X T L N X X S P X 271 A X K T F E K N S X A K N X G Q S L R D V L M X Y K X X G Q 301 X H X X X A X D F X A A N V E N S S Y P A K I Q K L P H F D 331 L R X X X D L F X G D Q G I A X K T X M K X V V R R X L F L 361 I A A Y A F R L V V C X I X A I C Q K K G Y S S G H I A A X 391 G S X R D Y S G F S X N S A T X N X N I Y G W P Q S A X X S 421 K P I X I T P A I D G E G A A X X V I X S I A S S Q X X X A 451 X X S A X X A Single letter code for amino acids, also a three letter code. Refer to your genetic code handout. Levels of Protein Structure Primary Structure Amino acids combine to form a chain Each acid is linked by a peptide bond Io structure by itself does not provide a lot of information. Protein Structure II0 (secondary) structure Based on local interactions between amino acids Common repeating structures found in proteins. Two types: alpha-helix and beta-pleated sheet. In an alpha-helix the polypeptide main chain makes up the central structure, and the side chains extend out and away from the helix. The CO group of one amino acid (n) is hydrogen bonded to the NH group of the amino acid four residues away (n +4). Can predict regions of secondary structure Ribbon Diagram α-helical regions Beta sheet Two types parallel and anti-parallel Beta Sheet ribbon diagram antiparallel parallel Protein Structure III0 (tertiary structure) Complete 3-D structure of protein (single polypeptide) hexokinase Chymotrypsin with inhibitor Protein Structure IV0 (quaternary) structure Not all proteins have IV0 structure Only if they are made of multiple polypeptide chains Protein Explorer Web site that will show a model of a protein who’s structure has been solved. http://molvis.sdsc.edu/protexpl/frntdoor.htm 3-D structures must be experimentally determined through the technique of X-ray crystalography. Scientists are working on computer predictions of 3D structure but so far not much progress. Nucleic Acids DNA –deoxyribonucleic acid RNA –ribonucleic acid Polymer of deoxyribonucleotide triphosphate (dNTP) 4 types of dNTP (ATP, CTP, TTP, GTP) All made of a base + sugar + triphosphate Polymer of ribonucleotide triphosphates (NTP) 4 types of NTP (ATP, CTP, UTP, GTP) All made of a base + sugar + triphosphate So what’s the difference? The sugar (ribose vs. deoxyribose) and one base (UTP vs. TTP) Function Nucleic Acids Information Storage Information transfer / Recognition DNA / mRNA rRNA / tRNA / snRNA Regulatory microRNA ? DNA Information for all proteins stored in DNA in the form of chromosomes or plasmids. Chromosomes (both circular and linear) consist of two strands of DNA wrapped together in a left handed helix. The strands of the helix are held together by hydrogen bonds between the individual bases. The “outside” of the helix consists of sugar and phosphate groups, giving the DNA molecule a negative charge. Complimentary Base Pairs A-T Base pairing G-C Base Pairing DNA Structure The DNA helix is “anti-parallel” Each strand of the helix has a 5’ (5 prime) end and a 3’ (3 prime) end. DNA Structure 5 ‘ end Strand 2 (Crick strand) 3’ end Strand 1 (Watson strand) 5’end 3 ‘ end DNA Structure 1 61 121 181 241 301 361 421 atgatgagtg gcacaggaaa cgtttcctcg atgctccaca gctatagcgc caacatacag cacaacgatg gctctccgga cttggattta ctagaatcag aattactgga tattgctctg ctcaactctg ggtcctctct gcaagaccct ggtttattga gtctgaacca agagaaaatg ataacagcag gtactactac accaggtaag gaagatgaag gggagctcag ggatgacatc gcatctttgc aaggattgct tgatcgacac gttcaatttg gcagaaagct acctctgagg acgccatacg cgaatccact ggattttatc aacattaacc cgcagtccct tccattgtct ctagaaatta ttgggttgcc gaaggtttct agggtggaaa ctcagatgaa gctgagtttt cggattagaa acgcacatgc aagaaaaaac ttctttattc atctgccctc tgattgtata Because of the base pairing rules, if we know one strand we also know what the other strand is. Convention is to right from 5’ to 3’ with 5’ on the left. Chromosomes and Plasmids Chromosomes are composed of DNA and proteins. Proteins (histone & histone like proteins) serve a structural role to compact the chromosome. Chromosomes can be circular, or linear. Both types contain an antiparallel double helix! Genes are regions within a chromosome. Like words within a sentence. For an animation of the organization of a human chromosome see: http://www.dnalc.org/ddnalc/resources/chr11a.html Region (red box) of chromosome XI from the bakers yeast S. cerevisiae. Red and Blue colored boxes are genes (ORF). Note that either strand may encode a gene, but that all genes start at the 5’ end and finish at the 3’ end. http://www.yeastgenome.org/ RNA Almost all single stranded (exception is RNAi). In some RNA molecules (tRNA) many of the bases are modified (i.e. psudouridine). Has capacity for enzymatic function. One school of thought holds that early organisms were based on RNA instead of DNA (RNA world). RNA Several different “types” which reflect different functions mRNA (messenger RNA) tRNA (transfer RNA) rRNA (ribosomal RNA) snRNA (small nuclear RNA) RNAi (RNA interference) RNA function mRNA – transfers information from DNA to ribosome (site where proteins are made) tRNA – “decodes” genetic code in mRNA, inserts correct A.A. in response to genetic code. rRNA-structural component of ribosome snRNA-involved in processing of mRNA RNAi-double stranded RNA, may be component of antiviral defense mechanism. RNA A - hairpin loop B- internal loop C- bulge loop D- multibranched loop E- stem F- pseudoknot Complex secondary structures can form in linear molecule mRNA Produced by RNA polymerase as product of transcription Provides a copy of gene sequence (ORF) for use in translation (protein synthesis). Transcriptional regulation is major regulatory point Processing of RNA transcripts occurs in eukaryotes Splicing, capping, poly A addition In prokaryotes coupled transcription and translation can occur