Biochemistry Exam 1

Trace Elements Purposes

Trace Elements Purposes

Many aid as catalyst for biochemical reactions

Carbon

Most stable bonds

Abundant element

Can form bonds with up to 4 other atoms

Enthalpy

Making and breaking covalent bonds or ionic interactions

Entropy

Changes in organization, disorganization, chaos

ΔG

Determines the speed at which equilibrium is approached

Thermodynamics

Tells us a reaction should proceed if the products are more stable than the reactants

Kinetics

Tells us how fast the reaction will go, though doesn’t tell us anything about the final state of things

-ΔH and +ΔS

-ΔG

Spontaneous at all temperatures

+ΔH and -ΔS

+ΔG

Nonspontaneous at all temperatures

Nucleophile

Attacks molecule for its proton (H)

Electrophile

Gives proton (H) to other molecule

Polarity

Unequal sharing of electrons in covalent bonds

Hydrophobic

Nonpolar molecules

Hydrophilic

Polar molecules

Non-polarity

Equal sharing of electrons in covalent bonds

Amphipathic

Polar and nonpolar parts in the same molecule

Electrostatic Interactions

Charges interacting with each other

Charge can interact with partial or full charges

Decrease in strength when distance increases

Van der Waals

Induces dipole-dipole interaction

Very weak compared to charge or polar forces

Hydrogen Bonds

Part electrostatic, part covalent

Donor Hydrogen is the H in polar covalent bond (X-H)

C-H bonds cannot be donor hydrogens because they are nonpolar

Strongest Bond Angle

180°

Hydrophobic Effect

Strength of hydrophobic effect depends on surface area

Clustered lipids to form bigger clathrate cage is stronger than individual hydrophobic lipids

Entropy is increased with clustered hydrophobic lipids

Clathrate Cage

Highly ordered H2O molecules formed around a hydrophobic molecule

Self-Ionization of Water

H2O disassociates and reacts with itself

Bronsted-Lowry Acid

Proton Donors

Bronsted-Lowry Base

Proton Acceptors

pka low and the protons go

pka high and they stand by

When pka = pH

Concentration of acid and base are the same

Buffers

Resists the change in pH when additional acid or base is added

Amino Acid Stereochemistry

Every amino acid is in the L-stereoisomer configuration

Gibbs Free Energy Equation

Finding ΔG from only products and reactants

pH Equation

pH = -log10[H+]

Henderson-Hasselbalch Equation

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

Fischer Projections

Carbon backbone is vertical with most oxidized carbon at the top

Vertical bonds go away from you

Horizontal bonds reach out and hug you

Hydrolysis

Adding H2O to break a peptide bond

Thermodynamically favorable

Must be catalyzed to break peptide bond

Condensation

Removing H2O to form a peptide bond

Peptide Bond

Using a ribosome to make an NH2 makes the NH2 nucleophilic attack the C to make a peptide bond

Has resonance

Rigid and planar

Trans conformation

Central Dogma

DNA is transcribed into RNA, which is translated by ribosomes into amino acids that then code into proteins

Amino Terminal End

NH3+ end of peptide chain

Synthesize from amino terminal to carboxyl terminal

Carboxyl Terminal End

COO- end of peptide chain

Protein Purification

To understand the properties of individual proteins

Analytical Techniques

Usually takes a small amount of protein that is modified or destroyed in analysis

Preparative Techniques

Produces a large amount of protein and maintains native protein activity

Precipitation

After homogenization of material and removal of insoluble components, differential retention of proteins and other components in a soluble or insoluble phase

Solubility

Affected by pH or ionic strength of solution

pI = 0 when proteins are aggregated (positive charges are equally connected to negative charges)

Salting In

Proteins are insoluble at low ionic strength

Salting Out

Proteins are insoluble at high ionic strength

Column Chromatography

Differential retention of proteins and other components of cells using size exclusion, ion exchange, or affinity methods

Size Exclusion Chromatography

Using discs with holes to separate proteins by size

Largest proteins should sink to the bottom

Smallest proteins should stick to the top discs

Affinity Based Chromatography

Using chemicals to separate proteins of interest

Ion Exchange Chromatography

Using discs that are negatively charged to separate proteins by charge

Negative charges should sink to the bottom

Positive charges should stick to the top discs

SDS-PAGE Gel Electrophoresis

Denatured proteins are run through an electrically charged gel

Must break disulfide bonds to have a linear polypeptide chain

Smaller, faster proteins will run along the gel further than other proteins

Two Dimensional Electrophoresis

Columnar tube of decreasing pH

Broken disulfide polypeptides are dropped into tube with decreasing pH

Sanger’s Method

Used to determine identity of N-terminal residue

Edman Degradation

Used to sequence 20-30 N-terminal residues

Mass Spectrometry

Modern method for sequencing proteins and characterizing complex mixtures

Peptide Backbone

C and N make up backbone

Steric constraints on protein backbone angles

Trans conformation, bond angle = 180°

Ramachandran Plot

Map of allowed conformations

Pro and Gly side chains give off different conformations and plots from other amino acids

Secondary Structures

Repeating patterns of backbones

Must satisfy all H-bonds

Alpha Helix

Backbone carbonyl of amino acid forms H-bond to amine

Nearly all are right handed

Rise Per Turn Equation

p = n * d

d = 1.5

Antiparallel Beta Sheet

Side chains are on alternating sides

Interact favorably

H-bonds are 180°

Parallel Beta Sheet

Side chains are on same side

Width between H-bonded side chains = 4

Length between R Groups on same side = 7

Beta Turns

Gly and Pro chains are in this conformation because they are in cis

Fibrous Protein Structure

Repeating protein structures

All secondary structures

Keratin

Fibrous

Rich in AVLIMF

Nonpolar and hydrophobic

Coiled coil

Right handed

Coiled Coil

Left handed

Reduces overall turns in each alpha helix to 3.5 (originally 3.6) residues per turn

Leads to a repeating pattern of hydrophobic side chains

Collagen

Triple helical cable

Repeats of Gly and Pro

Fibroin Protein

Aligned, stacked, packed antiparallel beta sheets

Gly and Ala

Knobs and holes of Gly and Ala align

Energetics of Protein Folding

Disfavorable entropy (-ΔS) change due to many possible conformations (+ΔG)

Favorable entropy (+ΔS) change due to hydrophobic effect (-ΔG)

Favorable enthalpy (+ΔH) change due to stronger H-bonds (-ΔG)

Hydrophobic Core

Most proteins have hydrophobic, nonpolar R Groups LIVMF

Optimizes density and packing

Large negative effects for hydrophobicity on surface

Hydrophilic Surface

Most proteins have hydrophilic, polar R Groups RHKDE

Large negative effects for hydrophilicity in core

Side Chain Hydrogen and Salt Bridge Bonding

When hydrophilic surface is in the core

Buried H-bonds don’t give full benefits because they were already partially satisfied by water in unfolded state

Buried salt bridges are stronger, more likely to bond to each other than to H2O

Domain Tertiary Pattern

Independently folding segment of protein sequence

Motif Tertiary Pattern

Commonly recurring structural element found in domains with different overall folds

Quaternary Structure

Interactions between separately folded protein subunits to form an overall complex

Stabilized by non-covalent interactions or disulfide bonds

May include one or several polypeptides

Levinthal’s Paradox

10^143 conformations for a 100 amino acid protein

There is not enough history in the universe to randomly sample every combo of amino acids to find a native

Denature a Protein

Heat

Chemical Denaturants

Pathway Dependent Folding

Steps must be taken before protein refolds to native state

Heat and chemical denaturation could interrupt beginning steps of protein folding

Energy Landscapes

Proteins follow favorable paths in conformation space that funnel them towards a native state

Ideally, protein only has a few errors and step backs to get to native state

In reality, protein takes many steps and errors to get to native state, may not reach it at all

Disulfide Isomerases

Speed up exchange of incorrect with correct disulfide bonds to get to native state

Prolyl Cis-Trans Isomerases

Flip proline between cis and trans isomers to affect backbone to get protein to native state

Molecular Chaperones

Uses ATP to bind misfolded and partially unfolded proteins

Misfolding Diseases

Ultra stable fibers and aggregates are formed that are more stable than native state

Form amyloid plaques that cause neurodegeneration