Intro to Cell & Molecular Biology• How do we study cell biology?

– Reductionist view• Cells as tiny complex machines• Sum of parts = whole

• Your goal:– be able to explain the roles

various molecular parts play in cell biological processes

– With the same clarity as with macroscopic items (bicycles, stoves, trains, etc…)

Intro to Cell & Molecular Biology• How do we study cell biology?

– Parsimony• the simplest explanation for all

relevant data is preferred over more complex explanations

• The most parsimonious answer is not necessarily perfectly correct

• More data could make us revise it

Chemical basis: Bonding• Covalent Bonds: sharing of e-

– One pair shared = single bond C C– Two pairs = double bond C C– Three pairs = triple bond C C

• Electronegativity (EN) is the ability of an atom to attract electrons to itself– C = 2.5 N = 3.0 O = 3.5 H = 2.1 S = 2.6– Sharing is unequal between different atoms in a molecule

• Polar molecules have significant EN differences– H2O, CH3COOH

• Nonpolar molecules have little EN differences– CH3(CH2)nCH3

• Amphipathic molecules have different EN characteristics at different positions

– CH3(CH2)nCOOH

Chemical basis: Bonding• Noncovalent Bonds: attractive forces between atoms of

opposite charge– Ionic: fully charged Na+ Cl-

• Strength dependent on environment (salt crystal vs aqueous)– Hydrogen: partial charge (polar molecules)

Noncovalent bonding• Noncovalent Bonds: continued…

– Van der Waals: transient dipole interactions

– Hydrophobic: water fearing– Hydrophilic: water loving

Robot Lizards ExploitVan der Waals contacts

• Based on the Gecko• Adhesion depends on close

contact between surfaces “StickyBot”

• Video link

H2O• Can form 4 hydrogen bonds

– High energy barrier to liquid --> gas phase transition

• Highly polarized– Asymmetric structure - both H atoms on one side– Can dissolve many compounds

H2O• Can dissolve many compounds

– Acids: can release H+– Bases: can accept H+

pH = - log [H+]• Pure H2O pH = 7 , [H+] = [OH-] = 10-7 M

• Why are reactions so pH sensitive?– Amino acid functional groups can change state based on pH

Carbon• Central to the chemistry of life.

– Can form four covalent bonds, with itself or other atoms.

– Carbon-containing molecules produced by living organisms are called biochemicals.

Chirality and Stereoisomerism• Chirality and Stereoisomerism:

– Asymmetric carbons bond to four different groups.

– Two mirror-image configurations:• Enantiomers, (aka)• Stereoisomers

– Can be either D- or L-isomers

– Natural amino acids = almost all L-isomers

– Natural carbohydrates = almost all D-isomers

Classes of molecules• Miscellaneous co-factors

– Vitamins, ATP, NADPH, etc• Metabolic intermediates

– Glycolysis, TCA cycle, etc• Monomers

– Amino acids– rNTPs = A, G, C, U– dNTPs = A, G, C, T– Sugars

• Macromolecules

Classes of molecules• Macromolecules

– Lipids• Fats = glycerol esterified with 3 fatty acids

– Saturated, unsaturated, cis, trans• Phospholipids = glycerol + 2 fatty acids + 1 phosphate• Steroids = cholesterol and derivatives

Classes of molecules• Macromolecules

– Lipids• Fats = glycerol esterified with 3 fatty acids

– Saturated, unsaturated, cis, trans• Phospholipids = glycerol + 2 fatty acids + 1 phosphate• Steroids = cholesterol and derivatives

Classes of molecules• Macromolecules

– Lipids• Fats = glycerol esterified with 3 fatty acids

– Saturated, unsaturated, cis, trans• Phospholipids = glycerol + 2 fatty acids + 1 phosphate• Steroids = cholesterol and derivatives

Classes of molecules• Macromolecules

– Lipids• Fats = glycerol esterified with 3 fatty acids

– Saturated, unsaturated, cis, trans• Phospholipids = glycerol + 2 fatty acids + 1 phosphate• Steroids = cholesterol and derivatives

Monomers and polymers

Classes of molecules• Macromolecules

– Carbohydrates• ( CH2O )n• At n ≥ 5 self-reaction to form rings

– C5 = ribose monomer– C6 = glucose monomer

Classes of molecules• Macromolecules

– “Nutritional” sugars:» Glycogen = branched alpha 1-4 linkage, dense granules

in cell cytoplasm in animals» Starch = helical and branched alpha 1-4 linkage, within

membrane bound plastids in plants

plastid

Classes of molecules• Macromolecules

– “Structural” sugars:» Cellulose = long and unbranched, beta 1-4 linkage,

resist tensile (pulling) forces, plants» Chitin = unbranched, N-acetylglucosamine,

invertebrates» Glycosaminoglycans = components of extracellular

matrix for cartilage and bone, repeating (A-B)n structure

Classes of molecules• Macromolecules

– Nucleic Acids• Nucleotide monomers (rNTPs, dNTPs)• Storage and transmission of genetic information

– Phosphate + 5C ribose sugar + nitrogenous base

RNA DNA

H

• DNA is usually double stranded• RNA is usually single stranded

– RNA may fold back on itself to form complex 3D structures, as in ribosomes.

– RNA may have catalytic activity; such RNA enzymes are called ribozymes.

– Adenosine triphosphate (ATP) is a nucleotide that plays a key role in cellular metabolism

– Guanosine triphosphate (GTP) serves as a switch to turn on some proteins.

Classes of molecules

DNA is a useful reference-frame for sizes

Classes of molecules• Macromolecules

– Proteins• Amino acid monomers• Peptide bond formation• N-terminus versus C-terminus• Backbone is common, side chains (R) differ

Classes of molecules• Macromolecules

– Proteins• Backbone is common, side chains differ

– 4 categories of amino acid side chains» Polar charged D, E, K, R, H» Polar uncharged» Nonpolar» Unique

Classes of molecules• Macromolecules

– Proteins• Backbone is common, side chains differ

– 4 categories of amino acid side chains» Polar charged D, E, K, R, H» Polar uncharged S, T, Q, N, Y» Nonpolar» Unique

Post-translational modifications: Phosphorylation of –OH groups

Classes of molecules• Macromolecules

– Proteins• Backbone is common, side chains differ

– 4 categories of amino acid side chains» Polar charged D, E, K, R, H» Polar uncharged S, T, Q, N, Y» Nonpolar A, V, L, I, M, F, W» Unique

Classes of molecules• Macromolecules

– Proteins• Backbone is common, side chains differ

– 4 categories of amino acid side chains» Polar charged D, E, K, R, H» Polar uncharged S, T, Q, N, Y» Nonpolar A, V, L, I, M, F, W» Unique G, C, P

Hydrophobic and hydrophilic amino acid residues in the protein cytochrome c

Levels of protein structure• Primary

– Sequence of the polypeptide chain

H3N-MQWERTYIHAHAPKLCVN-COOHH3N-Met Gln Trp Glu Arg Thr Tyr Ile…

H3N-Methionine Glutamine Tryptophan…

Levels of protein structure• Secondary

– Alpha-helix (collagen)– Beta-sheet (spider silk)– Side-chain dependence to which form is adopted but stabilization

comes from backbone - backbone hydrogen bonding interactions

Levels of protein structure• Tertiary

– Side-chain dependent and mediated packing of the secondary elements– Fibrous proteins = elongated, often structural roles– Globular = compact, often enzymes

Protein domains can be modular

• Protein Domains– Domains occur when

proteins are composed of two or more distinct regions.

– Each domain is a functional region

Protein structures can be dynamic

• Dynamic Changes within Proteins– May occur with

protein activity.

– Conformational changes are non-random movements triggered by various events (e.g. binding, chemical mods…)

Levels of protein structure• Quaternary

– Interactions between 2 or more distinct polypeptide chains

• Protein-Protein Interactions– Results from large-

scale studies can be presented in the form of a network.

– A list of potential interactions can elucidate unknown processes.

Disease• Sickle-Cell Anemia (SCA)• Painful• Life-threatening

periods of crisis• e.g. vaso-occlusive

crisis• block blood flow

in capillaries

Disease• Sickle-Cell Anemia (SCA)

• Hemoglobin is composed of four polypeptide chains• Two alpha-globin subunits + two beta-globin

subunits• SCA is caused by a single amino acid substitution

in beta-globin• E6V

Protein structure and folding

• Anfinsen RNase A experiment– Denature (unfold) protein in urea• Observed loss of activity

– Dialyze the urea away• Observed refolding• Regain of activity

Demonstrated structural information is inherent to protein sequence• Follow a folding pathway• Fold to the lowest energy state

Two alternate pathways for protein folding

Chaperones prevent mis-folding• Molecular Chaperones

– HSP70 during translation of nascent peptide• Binds exposed hydrophobic regions• Hydrolyzes ATP in a bind-release cycle

Hartl, et al (2011)

Chaperones prevent mis-folding• Molecular Chaperones

– HSP70 during translation of nascent peptide• Binds exposed hydrophobic regions• Hydrolyzes ATP in a bind-release cycle

– Chaperonins assist post-translation

Protein folding and Disease• CJD (Mad Cow) & Alzheimers Disease

– PrPC --> PrPSc --> plaque– APP --> Ab42 --> plaque