Chapter 1: Life

Strong Acids completely dissociate in water

Not an equilibrium

Strong Bases completely dissociate in water

Not an equilibrium

Strong acid + Strong Both = water + salt

Proteins, Carbohydrates, and lipids

Raw materials for anabolism

ATP for maintaining our bodies

It takes lots of food to keep us warm

Digestible

enzymes convert to complex to simple

transport to tissues

catabolism to generate ATP

Indigestible

cellulose

Low surface Area foods

Hydrolysis

Key Concepts

Mouth

Teeth

Chewing

Saliva

Antimicrobials

Enzymes that degrade or poison microbes

Proteins

Mucopolysaccharides

Enzymes

Amylase

pH

Enzymes

Stomach

Acid

Enzymes

Purpose

Homogenization

Digestion of Proteins

Proteins

Stable polymers folded into complex structures

Peptide bonds

Amides

Acidic Conditions

Secreted after meal is eaten

Stomach lining epithelium is protected from acid by mucins

Cells in stomach lining secrete HCl

Strong Acid

Completely dissociates

Acidifies chewed food entering stomach

Unfolds proteins

Activates pepsinogen to pepsin in stomach

Zymogen/Protease

Pepsinogen/Pepsin

Hydrolyze peptide bonds

Zymogen- Inactive precursor protease (Pepsinogen)

Protease- Enzyme that hydrolyses peptide bonds (Pepsin)

Activation by acid

Small Intestine

Neutralization

Zymogens

Digestion of Proteins

Pancreatic Enzymes

Digestion of Fats

Bile Acids

Liver

Pancreatic Lipase (+Colipase)

Adsorption of small molecules

Intestinal lining

Transporters

Liver

Harvesting

Enzymer

Bile Salts

Bile acid synethesis

Pancreas

Zymogens

Hormones

Synthesis of digestive enzymes

Triacyglycerol

Pancreatic Lipase

Proteins

Trypsinogen/ Trypsin

Chymotrypsinogen/ Chymotrypsin

Pro-elastase/ Elastase

Pro-peptidases/ Peptidases

Plants

Photosynthesis

Glucose

Starch

Ate and Digested

Anabolism

Catabolism

Animals

Work in Body

Muscle Contraction

Dysequilibrium

Coordinated synthesis and Degradation

Complex Molecules

Glycogen

Lipids

Polymers

Proteins

folded complex structures

3 or 4 degrees structures

Amino Acids

20 common acids

R= side chains

Peptide bonds

Amides

Nucleic Acids

Nucleotides

Ribophosphate backbone

Phosphodiester

Polysaccharides

Monosaccharides

Glycosidic bonds

hemiacetals and acetals

Lipids

Fatty Acids

Triacyglycerol

FAs esterified to glycerol

Storage in Adipose

Oils - TAGs in plants - unsaturated FAs

Lard - TAGs in animals - saturated FAs

Phosphoacylglycerol

Sphingolipids

Cholesterol

Water

Key to Interactions of
Biological Molecules

Polar, Dipole

Ice Floats

Good Solvent, Bad Solvent

Water as a Solvent

Noncovalent Bonds

Hydrogen Bonds

Ion-dipole Interactions

Van der Waals Interaction

Dipole-Dipole Interaction

Hydrophobic Interaction

Ionic Interaction

Membranes

Lipid barriers between
aqueous compartments

Fatty Acids

Phosphoglycerides

Glycerol

Fatty Acids

Phosphate

Selective Permeability

Membranes are Complex

Transporters

Receptors

Signaling Lipids

Fat Soluble Materials

Fluid Mosaic Model

Proteins

Integral

Peripheral

Compartmentalization
of functions

Biological Polymers

Macroscopic Motion

Muscle, Bone, Sinew

Microscopic Motion

Dynein

Tissue Specific Expression of Genes

Genomic DNA

20,000 to 24,000 genes

~3000 are expressed in all cells

Structural Hierarchy: Molecules to Cells

Level 1: Monomeric Units

DNA --> Nucleotides

Protein --> Amino Acids

Cellulose --> Sugars

Level 2: Macromolecule

Chromatin --> DNA

Plasma Membrane --> Protein

Cell Wall --> Cellulose

Level 3: Supramolecular Complex

Chromatin

Plasma Membrane

Cell wall

Level 4: The cell and its organelles

Central Dogma

Replication, Transcription, Translation

Cell Mitosis

Segregating subcellular structures
into daughter cells

Evolution

Imprecise Self-replication

High Specific Heat of water

Terrestrial Animals

Internal water buffers
extremes of temperature

Low Density of Ice

Ice cap on large or moving bodies of water

Water doesn't freeze through

Temperature of water below ice is
buffered from subzero air

Equilibria

Kinetics

Rate of Chemical Reaction

"How fast?"

Slow rate results from
high activation energy barrier

Catalysts: lower activation energy barrier

Transition State:

Distinct conformation

Unstable

Intermediate between
substrate and product

Activation Barrier

Determined by energy of
Transition State

Activation Energy (delta G+)

Energy required to overcome
the Activation Barrier

Enzymes Stabilize Transition
State conformation

Thermodynamics

Heat, work, and energy of chemical reactions

"How far?"

Equilibrium

Dynamic interconversion of products
and reactants

Free Energy (delta G)

Excess Energy that can be
used as work or heat

Metabolism

Anabolism requires energy input

Synthetic

Uses ATP and reduced coenzymes

Photosynthesis

Eat

Reductive

More bonds to Hydrogen

Catabolism

Oxidative

More bonds to Oxygen

Degradative

Makes ATP and reduced conenzymes

Difinition

Bond dissociation energy

Hydrogen Bonds

Linear is stronger Hydrogen Bond

Bent is weaker Hydrogen Bond

Ion-dipole Interactions

Solubility of NaCl

Water is dipole

Orients Hydrogen toward anions and Oxygen toward cations

Biomolecules

Ribophosphate backbone

Charged side chains and Nitrogen and Carbon termini

Like charges attract and opposite charges repel

Critical for folding and function of biological molecules

Ionic Interactions

Van der Waals Interactions

London Dispersion Factors

Favorable Interactions between hydrophobic molecules

Occur in the absence of water

Weak, local interactions between the fluffy, deformable electron clouds of hydrophobic groups

Always attractive interactions

Biomolecule

Fatty Acid

Micelle

Hydrophobic tails in center of the micelle interact with London Dispersion Forces

Globular Protein

Hydrophobic amino acids interact in center of proteins

Dipole-Dipole Interactions

Hydrophobic Interactions

Biomolecules

Water forms cages around hydrophobics (antiparallel orientation of water dipoles); Very LOW Entropy of solvent

Decrease in entropy of water drives the burial of hydrophobic surface area

Hydrophobic groups condense. Preleases trapped water to bulk

Micelle forms. Water released to bulk solvent

Micelle formation: High Entropy of solvent

a-carboxylate : 2

a-amino : 10

Aspartate : 4

Glutamate : 4

Histidine : 6

Lysine : 10

Arginine : 12

Glycine

Alanine

Proline

Valine

Leucine

Isoleucine

Methionine

Phenylalanine

Tyrosine

Tryptophan

Serine

Threonine

Cysteine

Asparagine

Glutamine

Basic

Lysine

Arginine

Histidine

Acidic

Aspartate

Glutamate

Covalent link between side chains

Centrifugation

Separate Organelles

Differential Centrifugation

Spins very fast and separates organelles

Keeps breaking up the materials so that they become soluble proteins

Left with pellets containing ribosomes and large macromolecules

Separate Mitochondria

Isopycnic Centrifugation

Takes a sample and the more dense component settles to the botton while the least dense component floats in the center

Separate Soluble from Insoluble

Soluble Proteins

Membrane Components

Chromatography

Size Exclusion Chromatography

Separates based on size

Porous particles

Small molecules explore internal space and are out of flow path

Large molecules are more often in flow path, so they elute first

No adhesion to the stationary phase

Exchanges buffer components between sample matrix and column buffer

Ion Exchange Chromatography

Anion Exchange

Anion stick to (+) charged column

Choose buffer so analyte is (-) charged

Equilibrate column then adsorb analyte to column

Wash off impurities (+) and neutral impurities

Elute analyte with High salt or basic conditions (deprotonates analyte)

Cation Exchange

Cations stick to (-) charged column

Choose buffer so analyte is (+) charged

Equilibrate column then adsorb analyte to column

Wash off impurities (-) and neutral impurities

Elute analyte with High salt or basic conditions (deprotonates analyte)

Affinity Chromatography

His Tag

Engineered into gene sequence

Ni 2+ is immobilized on column

Proteins are engineered to have a His6 sequence

Imidazole chelates Ni 2+ on column, so His-tagged protein sticks on column

Elution: High imidazole in elution buffer

PolyT

Purpose: mRNA purification

Immobilized PolyT

Elution: Free Thymine

Estimating the Molecular Weight of a Protein by Electrophoresis

Migration of ions in electric field

Migration depends on Charge and Size

Isoelectric Focusing

Isoelectric Point

Ampholytes establish stable pH gradient

Proteins migrate to pH=pI

X-Ray Crystallography

Proteins are crystallized into 3-D lattices

Crystals are blasted with X-rays

X-rays diffract off of electron clouds of C,N,S,O-
but usually not H

Diffraction pattern is used to construct a
3D arrangement of atoms

Advantages

Very High resolution - 1.5 A

Large proteins and protein complexes

Disadvantages

Proteins must crystallize; not aqueous structure

Structure is static

Crystal contacts can distort important regions of proteins

Solution conditions are not "native"

NMR Spectroscopy

Track nuclear spin on unpaired nuclei (1H, 13C (6 protons and 7 neuts), 19F (9 prots and 10 neuts))

In high strength magnetic field- unpaired nuclei process

Pulse of radio frequency resonant to precession frequency of nucleus: nucleus will flip to precess at different angle

After pulse- recover to original angle

Rate of recovery is related to the environment of the nucleus

Advantages

Protein is in solution

Motional dynamics of whole molecule

Dynamics of protein-ligand interactions

Disadvantages

Only small proteins can be studied

Complicated

No direct image of protein is obtained, just a series of spatical constraints

Circular Dichroism Spectroscopy

Differential absorption of circularly polarized light

Monitors protein structure by recognizing secondary structures

Chiral Centers

Unique absorption of circularly polarized light

Secondary structures are unique arrangements of chiral centers leading to unique spectra

Denaturation

Unfolding is cooperative

Monitor a signal

Spectral

Circular Dichroism

Fluorescense

Enthalpy

Denaturants

Heat

pH

Chemicals

Urea

Guanidine

Hydrophobic collapse drives the energetics of most protein folding

Fast process

Not random

Hierarchic process in which local secondary structures form first, followed by hydrophobic collapse

Molten globule by a spontaneous hydrophobic collapse first, followed by formation of secondary structures

Revised

"Native" structure is not always folded. Intrinsically Disordered Proteins!

Folded proteins may have many conformations. Proteins move!

Protein dynamics may play a crucial role in functions e.g. Histones!

Some proteins require chaperones to fold

They help overcome energy barriers or alter salvation to change the folding landscape

Changing primary structure can change the energetics of folded state or the folding energy landscape

Assume the native state is folded and is the most stable state

Noncovalent forces stabilize the native state relative to the unfolded state

Hydrogen bonding: Inter- and Intramolecular Hydrogen bonds

Ionic Interactions: Acidic and Basic groups (e.g. Asp-Lys)

London Dispersion Forces: Between hydrophobic residues in the protein interior

Hydrophobic Interactions: Drive burial of hydrophobics in the protein interior

Why is the Native state more stable?

Denatured state is less stable

Primary Structure

Amino Acid Residue

Primary Structure dictates fold...but it's complicated

Proteins start to fold before they are completely made

Other proteins can help fold the protein

Proteins may fold in one environment, then move to another environment

Ramachandran plot

Secondary Structure

Local Interactions

a- helix

Chain is coiled like a spring

Right-handed twist

R-groups protrude outwards

Peptide "backbone" is the core of the coil

Stabilized by intramolecular Hydrogen bonds between peptide bonds

Very compact structure

Geometry

All side chains point outward from core

There are 3.6 residues per turn- roughly every 3rd to 4th residue will be on same face

This allows for helix faces

Amphipathic: hydrophobic face and hydrophilic face

Charged: helix with a string of similarly charged residues

B- sheet

Extended peptide anions- pleated geometry

At Carbons are the apices

R-groups alternate (up, down, up, down..)

Planar peptide bonds are in the pleat

Peptide chains align side-by-side

Amide groups Hydrogen bond

Parallel and Antiparallel

Extended chains

B-turn

Type I occur 2x more frequently than Type II

Proline are preferred in 2nd position

Proline: conformationally restricted with fixed phi angle keeps turn rigid

Type II have Gly in 3rd position

Glycine: small side chain allows for tight corners

Hydrogen bond between the peptide bonds of 1st and 4th residues

Tertiary Structure

Polypeptide chain

Distinct Interactions

Independently Folding Regions (domains)

Quartenary Structure

Assembled subunits

Interactions between polypeptide chains

Not Globular

Long rope or sheet-like structures

Very tough- occur in skin, nails, cocoons

Hydrophilic, but not soluble

Structure of Hair

Keratin

Hair, Claws, Outer Epidermal Layer

Significant Cys content

Chemical Reduction of the Cystine

Crosslinking Keratin Fibrils: to curl or not to curl

Reduce --> Curl --> Oxidize

Structure of Silk

The fibers in silk cloth and in a spider web are made up of the protein fibroin

Fibroin consists of layers of antiparallel B-sheets rich in Ala and Gly residues

Structure of Collagen

Skin, Tendons, Cartilage, Bones

Significant Gly, Pro content