Lecture 2: Eukaryotic cells, amino acids

1. Lecture 2: Eukaryotic cells, amino acids • Prokaryotic cell features • Eukaryotic cell types • Amino acids • Quiz next Wed. on Amino Acids-you need to know the structures, names, single letter symbols, and pKas of the functional groups. You will have 30 min to take this quiz.

2. General schematic for a prokaryote cell

3. Prokaryotic cell structures • Cytoplasmic membrane/Inner membrane • About 45% lipid and 55% protein, forming a bilayer • Similar in structure and composition to the eukaryotic plasma membrane and mitochondrial membrane. • Highly selective pemeability barrier, transport-facilitated diffusion, active transport, group translocation • Electron transport and oxidative phosphorylation • Energy production • Motility • Replication • Cytosol - not just water- 20% protein by weight • Site of intermediary metabolism and energy production • Precursors necessary for biosynthesis • Nucleoid - Chromosomal DNA • Haploid genome. • Smaller than eukaryotic chromosomes, encodes ~ 3500 genes • No nucleus or nuclear membrane • Ribosomes • About 15,000 ribosomes per cell. Sites of protein synthesis

4. Prokaryotic cell structures • Capsule (slime layer), K antigen-another layer on the outside of the cell. • Both gram-positive and gram-negative bacteria can make capsules • Polysaccharide (exception is Bacillus anthracis (anthrax) which makes a poly-glutamate capsule). • Virulence-inhibits complement and phagocytosis • Glycocalyx-extracellular polysaccharide; biofilms; not a capsule • Flagella (H-antigen) for motility and chemotaxis • Pili (fimbriae) - Hair-like; protein • Adherence • Genetic exchange (fimbriae) • Fibrillar layer (protein coat, virulence) • Spores (Gram-positive bacteria only) • Metabolically inactive, resistant to heat (boiling), dessication, formed inresponse to stress • Storage granules - Storage forms of polymerized metabolites • Poly-3-hydroxybutyrate • sugars • Plasmids - non-chromosomal DNA • Usually circular, independently replicating, can be transmissible between cells by genetic exchange (conjugation), can encode antibiotic resistance

5. Archaea • Discovered as third or ‘intermediate’ branch of the tree by Carl Woese • Equidistantly related to both bacteria and eukaryotes (Eukarya) • Three main groups of Archaea • Methanogens - obligate anaerobes that produce methane (marsh gas) by reducing CO2 with H2. • Halobacteria - live only in concentrated brine solutions (>2M) • Thermoacidophiles - inhabit acidic hot springs • Many others found (~40% of ocean microorganisms!)

6. The Tree of Life has 3 primary branches: Bacteria, Archaea, and Eukaryotes

7. Eukaryotes • Larger than prokaryotic cells (10-100 µm) • Structural organization more complex than prokaryotes • Cell volumes 103-104 times larger than prokaryotic cells • Compartmentalization • Membrane bound nucleus • Internal membranes differentiated into specialized structures • Endoplasmic reticulum (ER) • Golgi apparatus • Animal vs. Plant cells

8. General schematic of an animal cell

9. Eukaryotic animal cell structures • Cell membrane/plasma membrane • About 50% lipid and 50% protein, ~5 nm thick • Similar in structure and composition to the prokaryotic inner membrane and highly selective pemeability barrier. • Pumps and channels • Enzymes • Reception of extracellular information • Nucleus- Separated from cytosol by nuclear envelope (double membrane) • DNA is complexed with histones (basic proteins) forming chromatin fibers • DNA organized into chromosomes • Nucleolus- Distinct region in the nucleus • Synthesizes rRNA • Site of ribosome assembly • Endoplasmic reticulum (ER) and ribosomes • Most extensive membrane in the cell • ER studded with ribosomes called rough ER • Synthesis of proteins and membranes

10. Eukaryotic animal cell structures • Golgi apparatus - Packages and processes macromolecules • Secretion • Delivery to other cellular compartments • Mitochondria - Separated from cytosol by double membrane • Markedly different in protein and lipid composition compared to the rest of the cell • 1 µm ~ same size of bacteria • Inner membrane and matrix contain many enzymes for energy metabolism. • Carbohydrates, fats, and amino acids are oxidized to CO2 and H2O. • Energy is converted to high-energy phosphate bonds (ATP) • Lysosomes - Intracellular digestion of materials • 0.2 - 0.5 µm single-membrane bounded vessicle • Contain hydrolytic enzymes such as proteases and nucleases • Formed from Golgi apparatus • Degrade cellular constituents targeted for destruction • Peroxisomes • 0.2 - 0.5 µm single-membrane bounded vessicle • Contain oxidative enzymes that use molecular oxygen and generate peroxides • Formed from smooth ER

11. General schematic of a plant cell

12. Plant cell structures • Cell wall • Cellulose fibers embedded in a polysaccharide/protein matix • >0.1 µm thick • Rigid and porous to small molecules. • Cell membrane - similar to animal cell membrane • ER, Golgi apparatus, ribosomes, lysosomes, peroxisomes • Similar to animal cells • Chloroplasts - site of photosynthesis • Light energy is converted to chemical energy (ATP) • Double membrane, inner volume is called stroma • Rich in membrane and encloses the thylakoid lumen • Photosynthetic reactions take place on the thylakoid membranes • Formation of carbohydrate from CO2 takes place in stroma • Much larger than mitochondria • Mitochondria • Similar to animal cells, responsible for energy generation in the dark • Vacuole - functions in transport and storage of nutrients andd cellular waste products • Most obvious part of plant cell • Enclosed by a single membrane called the tonoplast

13. Amino acids • The origins of biochemistry are tied to protein research • Why proteins? • Amino acids-the building blocks of proteins • The most well-defined physicochemical properties • Easier to isolate and characterize than nucleic acids, polysaccharides, or lipids • Proteins had easily recognizable functions (enzymes) • Amino acids • The building blocks of proteins • Diversity of function in proteins arises from the intrinsic properties of only 20 commonly occurring amino acids. • Can be polymerized • Novel acid-base properties • Varied structure and chemical functionality in amino acid side chains • Chirality

14. R Side chain R H - + C H COO H3N Carboxyl group Amino group COO- NH3 Amino acids are tetrahedral structures General structure for amino acids Except for proline, all amino acids possess the following structure: C

15. C H H H - + COO H3N R R C C - + COOH COO H3N H2N Ionic forms of amino acids R H+ H+ Zwitterion pH 7 Net charge 0 pH 1 Net charge +1 pH 13 Net charge -1

16. Amino acids can join via peptide bonds • The amino (-NH3+) and carboxyl (-COO-) groups allow amino acids to polymerize. • Amino acids react in a head-to-tail fashion • Elimination of a water molecules • Formation of covalent amide linkage (peptide bond) • Peptide bond formation is thermodynamically unfavorable-so reaction is coupled • Peptides • 2 amino acid (aa) residues - dipeptide • 3 aa residues - tripeptide • A few aa residues - oligopeptide • Many aa residues - polypeptide • Proteins are molecules that consist of one or more polypeptide chains • Polypeptides range from 40 to 33,000 amino acids (most about 1500 aa)

17. Condensation of two amino acids to form peptide bond

18. Polypeptides • Polypeptides are linear polymers • 20 standard amino acids • 3 main groups (note there are crossovers) based on R-groups (side chains) • Nonpolar (hydrophobic) side chains • Simple aliphatics • Aromatics • Proline • Uncharged polar side chains • Alcohols • Sulfur containing • Amides • Charged polar side chains • Acidic • Basic • From these 20 choices we get diversity • For a dipeptide, 202 = 400 possible dipeptides • For a tripeptide, 203 = 8000 possible tripeptides • For a 100 aa polypeptide, 20100 = 1.27 X 10130 possible polypeptides!

19. + + + + COO- COO- COO- COO- H3N H2N H3N H3N C C H H C C H H CH2 H CH2 CH3 CH2 CH2 CH2 Glycine Gly G Alanine Ala A S Proline Pro P CH3 Methionine Met M Aliphatic nonpolar amino acids: Gly, Ala, Met, Val, Leu, and Ile

20. + + + COO- COO- COO- H3N H3N H3N C H C C H H H C CH3 CH2 CH CH3 CH3 CH CH2 CH3 CH3 Valine Val V CH3 Leucine Leu L Isoleucine Ile I Aliphatic nonpolar amino acids: Gly, Ala, Met, Val, Leu, and Ile

21. + + + COO- COO- COO- H3N H3N H3N C C C H H H CH2 CH2 CH2 C CH NH Phenylalanine Phe F Tryptophan Trp W OH Tyrosine Tyr Y Aromatic nonpolar amino acids: Tyr, Trp and Phe

22. Amino acids • Nonpolar amino acids • All amino acids with alkyl R-groups (Ala, Val, Leu, and Ile) • Pro, Met, and Gly • Aromatic amino acids Phe, Tyr, and Trp • Generally hydrophobic • Exceptions are Pro, Gly, Tyr and Trp

23. + + + + COO- COO- COO- COO- H3N H3N H3N H3N C C H H C C H H H C OH H CH2 CH2 CH3 Glycine Gly G OH Serine Ser S Threonine Thr T OH Tyrosine Tyr Y Uncharged polar side chains

24. + H3N Uncharged polar side chains COO- C H R-S- + H+ R-SH CH2 SH R-O- + H+ R-OH Cysteine Cys C

25. Formation of cystine

26. + + COO- COO- H3N H3N C C H H CH2 CH2 C CH2 O NH2 C Asparagine Asn N O NH2 Glutamine Gln Q Uncharged polar side chains

27. Amino acids • Polar, uncharged amino acids • Contain R-groups that can form hydrogen bonds with water • Includes amino acids with alcohols in R-groups (Ser, Thr, Tyr) • Amide groups: Asn and Gln • Usually more soluble in water • Exception is Tyr (most insoluble at 0.453 g/L at 25 C) • Sulfhydryl group: Cys • Cys can form a disulfide bond (2 cysteines can make one cystine)

28. + + COO- COO- H3N H3N C C H H Charged polar (acidic) side chains CH2 CH2 CH2 C C O O- O O- Aspartic acid Asp D Glutamic acid Glu E

29. Amino acids • Acidic amino acids • Amino acids in which R-group contains a carboxyl group • Asp and Glu • Have a net negative charge at pH 7 (negatively charged pH > 3) • Negative charges play important roles • Metal-binding sites • Carboxyl groups may act as nucleophiles in enzymatic interactions • Electrostatic bonding interactions

30. + + + COO- COO- H3N H3N H3N C C H H Charged polar (basic) side chains COO- C H CH2 CH2 CH2 CH2 CH2 CH2 CH2 C HC NH CH2 NH H+N C NH3+ CH NH2 NH2+ Lysine Lys K Histidine His H Arginine Arg R

31. Amino acids • Basic amino acids • Amino acids in which R-group have net positive charges at pH 7 • His, Lys, and Arg • Lys and Arg are fully protonated at pH 7 • Participate in electrostatic interactions • His has a side chain pKa of 6.0 and is only 10% protonated at pH 7 • Because His has a pKa near neutral, it plays important roles as a proton donor or acceptor in many enzymes. • His containing peptides are important biological buffers

32. Nonstandard amino acids • 20 common amino acids programmed by genetic code • Nature often needs more variation • Nonstandard amino acids play a variety of roles: structural, antibiotics, signals, hormones, neurotransmitters, intermediates in metabolic cycles, etc. • Nonstandard amino acids are usually the result of modification of a standard amino acid after a polypeptide has been synthesized. • If you see the structure, could you tell where these nonstandard amino acids were derived from?

33. Nonstandard amino acids

34. Nonstandard amino acids