BCH4024 - Exam 1

Foundations in Biochem

• cellular

• chemical

• physical

• genetic

• evolution

Cellular Foundations

• cells are fundamental units of life

• at each level of organization novel properties emerge; life emerges at cellular level

Common characteristics of living things

• organization

• expression and transmission of genetic info

• transfer and transformation of energy and matter

• interact w/ their system and enviro

• evolution - conservation of function and diversity in structure

Phylogeny of Life

Bacteria

Archaea

Eukarya

Prok and Euks contain

• DNA

• ribosomes

• cytosol

• plasma membrane

euks contain

DNA in membrane bound nucleus and membrane bound organelles

Cells are limited by diffusion

• upper limit of cell size is set by rate of diffusion

• as size increase, surface to volume ration decreases

  • many animal cells have highly convoluted/folded surfaces

Cell Cytoskeleton

dynamic and made of protein filaments

• Actin filaments (smallest) - 6nm wide, made from G-actin

  • polymerizes to F-actin through noncovalent bonds

• Intermediate filaments (medium) - 10 nm wide, made from a-keratin subunits

• microtubules (largest) - 23nm wide, made from tubulin

Each filament composed of protein monomers that are bound NONcovalently

• provide shape, organization, and help things move

Nucleic Acids

DNA and RNA are polymers of nucleotides

• store and transmit genetic info

• some RNAs have structural and catalytic roles

Genome = entire seq of cell’s DNA or RNA

Genomics = characterization of structure, function, evolution, and mapping of genomes

Proteins

Long polymers of AAs

• function as enzymes, structural elements, signal receptors, transporters

Proteome = sum of all proteins functioning in a cell

Proteomics = systematic characterization of this prot complement under a specific set of cond

Polysaccharides

Polymers of simple sugars

• Energy rich fuel stores, rigid structural components, extracellular recognition elements

Glycome = entire complement of carbohydrate-containing molecules

Lipids

Water-insoluble hydrocarbon derivatives

• structural comps, energy-rich fuel stores, pigments, intracell signals

Lipidome = lipid containing molecules in a cell

Chemical Foundations

Less than 30 of naturally occurring elements essential to life

• H, O, N, C make up 99% of organisms

Enantiomers

non-superimposable mirror images

Physical Foundations

• molecules are consistently synthesized and broken down

• Living cells are in a dynamic steady state distant from eq with its surroundings

  • maitaining requires constant investment of energy

• Organisms either make energy or extract it (photoautotrophs vs chemotrophs)

Laws of Thermodynamics

1. total amount of energy in universe remains constant

2. randomness (S, entropy), in universe is constantly increasing

   1. creating and maintaining order requires work and energy

Free Energy

Want deltaG to be less than zero for a rxn to happen spontaneously

DeltaG = deltaH - TdeltaS

Keq > 1

prods > reactants

G < 0

rxn is spont

Keq < 1

prods < reactants

G > 0

rxn is NOT spont

Keq = 1

prods = reac

G = 0

rxn @ eq

standard free energy

G⁰ = -RTlnK

Actual free energy change

G = G⁰ + RTlnK

K = Q

rxn is at eq

G = 0

K < Q

rxn is past eq

more prods/reacts now then at eq

G > 0

rxn is not spont

K > Q

rxn has not reached eq

less prods/reacts now then at eq

G < 0

rxn is spont

H bonds are fleeting

1-20 ps

H bonds are ___ and ____ than covalent bonds

longer and weaker

23 kj/mol vs 470 kj/mol

liquid water has _ h bonds vs ice has _

3.4 vs 4

Weak noncovalent interactions compared to covalent

• Hydrophobic and aromatic ring stacking and van der waals (weak but many)

  • 0.4 - 4 kj/mol (weakest)

• H bonds and electrostatic

  • 4-40 kj/mol

• Salt bridge (H bond + electrostatic)

  • 40-400 kj/mol

  • carboxylate AA side chain (Asp, Glu) to basic AA (Arg, Lys)

Hydrophobic Effect

Water molecules forming cage around nonpolar substances and releases ordered water as it clusters together

• increases entropy

• amphipathic molecules form micelles

• similar interactions w/ enzymes and substrates

Kw of water

1.0 × 10^-14 M²

henderson-hasselbalch

pH = pKa + log [A-]/[HA]

Keq of water at 25C

1.8 × 10^-16 → can normally assume concentration is 55.5M

optimal pH for enzymes

have max catalytic activity

pepsin - pH of 1.5 (digestive enzyme)

Trypsin - neutral pH in lumen (digestive enzyme in SI)

Alkaline phosphatase - pH of 8-9 (hydrolytic enzyme of bone tissue)

Diabetes results in

acidosis

• accumulation of high conc of beta-hydroxybutyric acid and acetoacetic acid which lowers blood pH to less than 7.35

AA structure

Proteins consist almost entirely of what stereoisomer

L-amino acids

• rare D-amino acids but converted after prot syn

optically active due to chirality

Zwitterion

have both positive and negative charge at certain pH ranges

****MAJOR FORM

AAs also called ampholytes due to having + and - charge (acidic and basic groups)

Isoelectric point of AAs

where net charge EQUALS ZERO

pI = (pka1 + pka2) / 2

Glycine

Nonpolar, achiral, hydrophobic

G, Gly

Alanine

nonpolar,hydrophobic

A, Ala

Proline

nonpolar, hydrophobic

P, Pro

can be in cis or trans conformation

Valine

hydrophobic, nonpolar

V, Val

Leucine

Hydrophobic, nonpolar

L, Leu

Isoleucine

hydrophobic, nonpolar

I, Ile

Methionine

hydrophobic, nonpolar

M, Met

Pheylalanine

aromatic, rel hydrophobic

F, Phe

Tyrosine

aromatic, rel hydrophobic

Y, Tyr

can H bond, plays important functional role in some enzymes

Tryptophan

aromatic, rel hydrophobic

W, Trp

Serine

polar, uncharged

S, Ser

Threonine

polar, uncharged

T, Thr

Cysteine

polar, uncharged

can form cystine through disfulfide bonds → not under physiological pH (pka = 8.3)

C, Cys

Asparagine

polar, uncharged

N, Asn

Glutamine

polar, uncharged

Q, Gln

Lysine

positively charged, basic

K, Lys

Arginine

positively charged, basic

R, Arg

Histidine

positively charged, basic

H, His

only AA w/ pka near neutrality

• can be H+ donor or acceptor

Aspartate / Aspartic Acid

negatively charged, acidic

Asp, D

Glutamate / Glutamic Acid

negatively charged, acidic

Glu, E

Characterize biomolecules by light absorption

Absorbance of wavelengths

• aromatic AAs have distinct wavelengths

C labeling in AAs

Peptide Bond

• molecule of water eliminated for each peptide bond formed

• covalent bond

• amide linkage

• requires energy to form

• to break raise temp or use enzyme that lowers activation energy

Peptide Bond Structure

Linus Pauling and Robert Corey Discovered

• C-N bond is 10% shorter than usual amide bonds due to double bond character (40%) from resonance

• makes it stronger and shorter (1.32 A)

• planar

Peptide bond angles

phi = Ca-N

psi = Ca-C=O

omega = peptide bond

Peptide Nomenclature

N-terminus on the left and C-terminus on the right

• AAs are added onto C-terminus

Protein Size

avg MW of nucleotide is 110 Da

to estimate # residues in a protein take proteins MW and divide by AA MW

Number of Possible Prot Seqs

20^a

a = peptide length

• orgs typically rely on 30,000 - 50,000 seqs so necessary function will determine which sequence is constructed

Folding is directed by

AA side chains

Determine peptide sequence by

• Mass Spec

• Proteases

Conjugated proteins

permanently associated chemical components (prosthetic groups)

• lipoproteins

• glycoproteins

• metalloproteins

proteins are stabilized by

noncovalent interactions and forces

primary structure

backbone of protein, sequence of AA residues

• 3 covalent bonds separate alpha C of adjacent AA residues

• dihedral angles (phi and psi) define secondary strucs

Secondary Structures

spatial arrangement of main chain atoms

• alpha helix, beta conformation, b turn, random coils

• phi and psi remain same throughout a segment to give uniform structures

• stabilized by weak noncovalent interactions

• r groups ALWAYS protrude out from backbone

alpha helix

simplest arrange, max number of H bonds

• every 4 AAs: H of n and O of n+4 (of main chain backbone)

• each turn is 5.4A and 3.6 residues

• polar residues on one side and hydrophobic on the other

• Pro and Gly destabilize the helix

  • Pro introduces a kink and Gly allows too much flexibility

• Generally right-handed, left-handed is less stable

• small electric dipoles align through H bonds in each peptide bond (N → C)

3₁₀ = smallest, 3 residues

alpha = medium, 3.6 residues

pi = largest, 4.4 residues

Beta conformation

Backbone extends into zigzag (7 A), organizes polypep into sheets, R groups are trans

• antiparallel (most common due to linear H bonding between strands)

• Parallel (less common, distorted H-bonding)

  • forms between backbone atoms of adjacent strands

Beta turns

connect ends of two adjacent segments of an antiparallel B sheet

• 180 degree turn

Type 1 → 4 residues, proline is the 2nd residue

Type 2 → 4 residues, glycine is the 3rd residue

Gamma turns → 3 residues, proline is the 2nd residue

H bond forms btwn 1st and last residue

Ramachandran Plots

Visualizes phi and psi angles and tests quality of 3D protein structures

Circular Dichroism

• assesses common 2nd structures

• measures differences in molar absorption of left vs right-handed circularly polarized light

AAs found in alpha helix

Kristin Has Marvelous LACE Q tips

AAs found in Beta Sheet

IVY For the Win

AAs found in reverse turns

SPeeDiNG

What AA is found in all secondary stucture

Arginine

Tertiary Structure

overall 3D structure

• arrangement of all atoms in a prot

• weak interactions and covalent bonds hold interacting segments in position

Quarternary Structure

arrangement of TWO+ polypeptide chains in 3D complexes

Types of Proteins

• Fibrous

• Globular

• Membrane

• Intrinsically Disordered

Fibrous Proteins

• give strength/flexibility to structures

• simple repeating element of 2nd struc

• water insoluble due to high conc of hydrophobic residues

• alpha helix cross-linked with disulfide bonds

• beta sheets

• collagen triple helix

alpha-keratin

• two right-handed alpha helices wrapped around each other to form a super-twisted coil (overall left-handed_

• Common AAs: Angry Vikings Make Fantastic Iced Lattes

• cross-links stabilized by disulfide bonds

Collagen

found in connective tissue

• 3 left-handed alpha-helices twisted around each other right-handed

• rich in Gly-X-Y

  • Y = hydroxyproline

  • X = proline

• Cross-linked by covalent bonds involving Lys, hydroxylysine, histidine

  • super strong

Scurvy

general degradation of connective tissue

• lack of vitamin C

• Vit C required for hydroxylation of proline and lysine in collage

Globular Proteins

fold back on each other

• more compact

• distinct structure, unique for biological function

Myoglobin

Intrinsically Disordered Proteins

• lack defineable structure

• lack hydrophobic core

• high densities of charged residues

  • Every Kid Reeks of Poop

• facilitates a protein to interact w/ multiple binding partners

• disordered segments can assume diff structures

Example: p53

Proteostasis

Protein homeostasis: folding, unfolding, and degradation of proteins

Native proteins

synthesized from intermediates or chaperone assisted folding

Thermodynamics of Prot Folding

• Unfavorable entropy change (negative S)

  • folding decreases protein’s internal S

• Favorable enthalpy (negative H)

  • folding creates stable internal bonds → release heat

• Hydrophobic effect

  • increases S of water, MAIN driving force for folding

Denaturation

by heat, pH, organic solv, solutes, detergents, etc.

• lead to protein precipitation

• loss of function

• determine stability of prot by playing with one of these factors

Anfinsen Experiment

Showed that the primary struc (AA seq) contains all info needed to fold a protein chain into its tertiary struc

• renaturation

• Denatured RNase A w/ urea and mercaptoethanol (to break disulfide bonds)

• Removed solvents and RNase A became catalytically active again

Folding is a rapid stepwise process

• local secondary strucs fold first

  • ionic interaction and H bonds play important role

• longer range interactions follow

  • hydrophobic effect

• process continues until entire polypep folds

Levinthal’s Paradox

mathematically impossible for protein folding to occur randomly by trying every conformation until the lowest energy one is found

Free-Energy Funnel

multiple stable intermediates leading to final fold

• high degree of conformational entropy

• high free energy

Chaperone Proteins

facilitate correct folding pathways or ideal microenvironments