Exam One Content

All life on earth comes from the — via —

the sun via solar energy

Photosynthesis converts — into —

CO2 into carbohydrates and oxygen

ATP in all processes (order of conversion)

Solar energy —> chemical energy —> carbohydrate energy —> ATP energy → drives other work (chemical, transport, mechanical)

mechanical work takes — from — to —

ATP from chemical work (metabolic pathways) to have muscle contractions

First law of thermodynamics

in living systems, energy cannot be created or destroyed

energy can be converted from one form to another

Second law of thermodynamics

Every spontaneous process increases the total entropy of the universe

• tends towards disorder

Rank disorder from solids

Solids: lots of hydrogen bonding + tight packing + little movement → lowest entropy

Liquids: hydrogen bonds are breaking and forming

Gas: no hydrogen bonds forming → highest entropy

relate the conundrum between homeostasis + laws of thermodynamics

• Second Law: universe tends towards disorder

• Cells: very highly ordered system

  • To maintain homeostasis it requires an input of energy into the system (ATP)

the cell membrane is a — system where — move into the cell, and — move out of the cell via —.

Relate the conundrum to this.

Open, fats and sugars, waste materials, transport proteins.

maintaining homeostasis with an open system requires a large input of energy

Enthalpy: in a — we are talking about what happens with the —.

• describe endothermic/exothermic conditions

• Single to Double bonds

biological system, bonds

• exothermic: releasing energy via high energy bonds to low energy bonds = favorable

• Endothermic: using energy to break bonds = unfavorable

• going from single to double covalent bond is exothermic due to energy release

• Gibbs free energy equation

• Equation related Gibbs Free energy and equilibrium constant

• ΔG = ΔH - TΔS

• ΔG’° = -RT ln k’_eq

Standard Conditions

Pressure = 1 atm

Room temperature = 25°C, 298K

Concentration = 1M reactants and products

pH = 7

h2O = 55.5M

Mg²⁺ = 1mM

R = 8.315 J/Mol·K

Kelvin = °C + 273.15

pH Scale difference

ex) at 2 pH units will increase by —

10-fold difference (log scale)

2 pH = 100 times

Why is water polar

• electronegativity difference of oxygen

• Oxygen has 2 lone pairs, causing the shape + angle of H2O

• Shape will cause a dipole

Bond angle in water + bonds

104.3°

H-O-H held together by strong covalent bonds

Why are H-bonds weak and transient

they are constantly breaking and reforming which allows for maximum entropy to occur

• this is important for solvation properties

Requirements for a hydrogen bond to occur + how many hydrogen bonds can one water molecule form

• a dipole (S+ and S-)

• Need hydrogen donor (S+ on H) and acceptor (S- on O)

• Water molecules need to be close in proximity due to them being easily broken/reformed

• 4 H bonds possible per water molecule

Hydrogen bonds + water resistance to temperature

electrostatic interaction between hydrogen donor and acceptor

• H-bond network will absorb energy + redistribute the energy as the H-bonds are disrupted/rearranged

  • gives water a high specific heat

• not strong on its own, but strong when you have multiple

Ion-dipole and dipole-dipole interactions with water

Ion-dipole:

• Cation: dipole allows the (s-) oxygens to orient to surround the K+ and dissolve it

• Anion: (s+) hydrogens will surround Cl- and dissolve it in water

Dipole-Dipole: interactions of polar compounds with water

higher — of water will move around ions and — them in water

disorder/entropy, solvate/dissolve

What happens to water in hydrophobic interactions

• water becomes highly ordered due to formation of water cages

  • we want entropy to increase so fats will aggregate to reduce the nonpolar surface exposed to water → fewer ordered water molecules and waters entropy increases

• Micelles = all hydrophobic groups are isolated form water resulting in a less ordered shell of H2O, increasing entropy

pH and pKa equations

pH = -log[H+]

pKa = -log Ka

• Ka = acid dissociation constant = ability to ionize

pKa measures —

strength of the acid

• Adding strong acid: complete ionization and releases H+, decreasing pH

  • high Ka, low pKa

• Adding weak acid: lower tendency to ionize, affecting pH to less dramatic degree

  • Low Ka, high pKa

pKa is the — where an ionizable group is —

pH, 50% protonated and 50% deprotonated

• tells us the pH we are happy to give up a proton

Good buffer

Weak acids since they are completely reversible and will have a reserve of H+ to neutralize any bases that are added and a place to store protons as well

• can resist changes to the pH

inflection point

when pH = pKa

• [acid] = [base/salt]

• 50% of species in conjugate base form → ability to soak up any H+

• 50% of species in acidic form → reserve of H+ to neutralize bases

pKa

right int he center of the buffering range

Titration Curve → ionizable groups

Ionizable groups are pKa values and is where the curve flattens

ex) 3 flattenings of curves = 3 pKa = three buffering regions

Henderson Hasselbalch Equation + understanding + application to drugs

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

• determines the ratio of protonated to deprotonated species at a given pH

• uncharged/neutral species cross hydrophobic lipid bilayer better than charged. Henderson will predict how much charged/uncharged is present at a pH —> lets us see how drugs can pass through the body

Explain ATP Coupling

• ATP Hydrolysis: ΔG < 0 → very thermodynamically favorable

• couples with unfavorable reactions resulting in addition of ΔG values and overall processes becoming negative/spontaneous

ATP Structure

Adenine + Ribose + 3 Phosphates

• two high energy phosphoanhydride bonds connecting 3 phosphates together

• one phosphodiester bonds connecting phosphates to the ribose

What does it mean that ATP has a high phosphoryl-transfer potential

ATP readily transfers a phosphate group because the hydrolysis produces a more stable, lower free-energy product, giving it a large negative ΔG

What drives Hydrolysis

1. ATP has a -4 charge due to the phosphate groups

   1. hydrolysis will relief the charge repulsion

2. The phosphate exhibit a lot of resonance stabilization → delocalization of the negative charge on all oxygens → increases stability

ATP-ADP Cycle + conundrum

• Anabolic and catabolic processes are linked

1. Catabolism:

   1. metabolic fuel is broken down, releasing energy

   2. energy is captured to drive ADP + Pi into ATP

2. Anabolism: Building larger/complex molecules from smaller ones (biosynthesis) is unfavorable

   1. Couples with spontaneous, ATP hydrolysis to allow this reaction to proceed

   2. forms ADP + Pi

What does energy charge tell us, and how do ATP, ADP, and AMP change with energy charge?

X-axis: energy charge Y-axis: fraction of total nucleotide (ATP/ADP/AMP)

Physiological range = 0.7-0.9 → ATP is favored

Energy charge = energy status of ATP, ADP, AMP

• energy charge of 1: High ATP, low ADP and AMP

• Energy charge of 0: high AMP, low ADP and ATP (stored energy depleted)

ADP: highest intermediate energy charge

Primary Structure of a protein

• amino acids are joined via peptide bonds between carboxy and amino group of 2 amino acids

• Condensation reaction where H2O is released

  • OH from carboxy, and H from amino

After the polypeptide has formed, the primary structure has —

the information for folding within the sequence of amino acids

• determines how they interact to fold

final fold of a peptide is —

tertiary structure

peptide bonds can/cannot rotate + why + impact

the single covalent bond cannot rotate due to its partial double-bond character via resonance of the amide

• this rigidness results in the R groups alternating the sides they face

• this plays a big role in how R groups are available for folding

Driving force behind folding + Funnel Folding

• proteins want to reach minimal free energy state

• funnel folding relates to all folding intermediates being funneled into one final native form which is the minimal free energy state possible in the conditions of the medium

What can lead to an incorrect tertiary form?

If external forces affect local energy minima during the folding process, resulting in protein misfolding.

Markov State Model

illustrates the multiple intermediate folding shapes each individual polypeptide may take before reaching the final native form

Chaperones

If a protein gets stuck in a local minima (dip) on their way to the native state, chaperones, which are helpers of the cell, will aid in folding

• two types → clamps and chambers

What happens if chaperones are unable to correct the misfolding of proteins?

misfolded proteins may become amorphous aggregate, oligomersm incorrectly, or form large amyloid fibrils

amyloid fibrils

very rigid in structure and not able to be helped ack into their native state

aggregates

less rigid but are all tangled up together and unable to be separated to be correctly folded

Chamber Chaperonin Function

• provided a sequestered/isolated optimal environment to allow the polypeptide to fold

• important during cellular stress and when proteins are more usceptible to unfolding

• requires the use of ATP in their reaction cycle

Chamber Chaperonin Example

Bacterial GroES-GroEL

1. unfolded protein arrives: has exposed hydrophobic parts (bad since hydrophobic regions of multiple unfolded polypeptides can interact and aggregate)

   1. GroEL has hydrophobic binding sites around the rim of its open chamber which unfolded proteins bind to

2. ATP binds to GroEL: causes a conformational change, allowing the GroES to bind as a cap (cis)

3. protein folds: inside the closed cis chamber, the protein is separated from other proteins, and can fold without sticking to other unfolded proteins

4. ATP is Hydrolyzed: 7ATP —> 7ADP + 7Pi

   1. timer: gives the protein about 10 seconds to fold inside the chamber

5. Opposite (trans) end gets ready:

   1. As the protein folds in the cis chamber the trans ring is open

   2. another unfolded protein can bind on the open chamber, ATP can also bind to it

6. Trans gets capped:

   1. Binding of ATP can cause GroES to bind to the tras chamber, causing the old cis ring to open (newly formed folded protein exits here)

   2. This shows the asymmetrical reaction

7. Old protein exits:

   1. protein inside cis chamber is released, if correctly folded, it will leave as a native protein. If incorrectly folded, it can go through the process again.

Clamp Chaperone

At the ribosone, while we are undergoing translation, DnaJ and DnaK will bind to hydrophobic patches

• this will prevent the hydrophobic patches from collapsing on each other before we have all the information from the polypeptide

• The clamps will only released the polypeptide after the whole polypeptide is translated and released so that it has the information to fold properly

How much protein requires the help of chaperones?

• 70% of proteins outside of translation can fold on their own without chaperones

• 30% of proteins will need help from clamp proteins

  • usually large proteins with hydrophobic patches

  • 20% of proteins only need clamp proteins

  • 10% of proteins need clamps and chambers

Three models of protein folding

1. framework model

2. Hydrophobic collapse model

3. Nucleation-condensation model

Framework model

1. first forms the secondary structure with beta pleated sheets and alpha helices

2. will coalesce/come together to form a secondary structure

3. native form will come after this

Hydrophobic collapse model

Important in aqueous environments

1. polypeptide comes together to bury all hydrophobic amino acids away fro the aqueous environment and collapses in the middle

2. the secondary structure will occur once the hydrophobic amino acids are bured

3. the native form forms

Nucleation-Condensation model

occurs in larger proteins

1. local folding at one part of the protein before the rest of the protein folds

2. secondary structural elements will come together at one part while the rest is unfolded

   1. the secondary/tertiary forms in one area before the secondary structure forms on another side

3. Another nucleation event can occur in another area

4. they eventually will all collapse together

The secondary structure consists of

alpha helices, beta sheets, beta turns

Who proposed alpha helix+ what did he make his assumptions off of?

Linus Pauling. He only knew bond distances and the amount of flexibility in peptide bonds

• Flexibility in peptide bonds is lower than flexibility in other bonds

• X-ray crystallography tested + confirmed his proposal

What was Pauling's proposal?

for optimal h-bond distance to occur, the C=O of residue n hydrogen bonds with the N-H of residue n+4

• These 2 hydrogen bond need to shorten the distance between them, the optimal conformation for this was the twist

describe the optimal twist/alpha helix

tight helix with the R groups facing outward, allowing them to interact with the environment. Intrastrand hydrogen bonds stabilize the helix.

Does the alpha helix unspontaneously form?

it spontaneously forms due to optimal stability

• Alpha helix is twisted around a central pole (imaginary pole) and it is a very tight twist

how many residues are present per turn, and what is this called?

3.6 residues per turn, this is called a repeated structure

• Each coil will have 3.6 aa, they stack onto each other, and the r groups face outwards

What is the distance/step of each coil?

5.4 Å

explain sidedness of the alpha helix:

helps all hydrophobic sides come together and help reduce the interaction with water to create a more stable conformation

• While the hydrophobic aa end up coming together to form a hydrophobic patch in the 3° structure, this doesn’t mean that the hydrophobic aa are all in the sequence immediately preceding each other

Explain peptide bond dipoles being transmitted through the alpha helix

each peptide bond has a dipole which come together to form an overall helix dipole

approximately how many amino acids can an α-helix contain?

min = 5 aa, max = 50 aa

Describe beta strand structure

A β-strand is an extended polypeptide structure in which the R groups alternate above and below the backbone. Multiple β-strands can associate to form β-sheets

Explain how B strands can be far but end up near each other

B strands can be distant in the primary sequence but end up next to each other through folding

• Once folding brings them close enough, their backbones can form H-bonds and create B-sheets (secondary structure)

are B sheets flat or rigid

b sheets are flexible

who proposed the beta strands and what did he say

William Astbury said you can have hydrogen bonds between different areas of polypeptides. In β-sheets, neighboring β-strands are stabilized by interstrand hydrogen bonds.

Order of amino acid sequence comes from — via —

DNA sequence via DNA → mRNA → protein via transcription and translation

not all — turn into — and are not all —

not all proteins turn into a secondary structure and are not all structural components

Determining secondary structures on diagrams

thin lines = unfolded proteins

thicker lines = secondary component

Tertiary Structure interactions

R group interactions

• while different folding paths may occur, they all end in the same final form due to them having the same information and same primary sequence

Quaternary Structure

more than one polypeptide, they come together to form larger assemblies

Gramacidine

• b-helix supersecondary structure

• buried in phospholipid bilayer

• good antibiotic when there as it provides a channel to affect the movement across the membrane, causing bacteria to die

• R groups are facing out from the structure and will be hydrophobic so they can be buried in the mmebrnae

• amphipathic/hydrophilic R groups would be between the interface of the hydrophobic tails and hydrophilic heads

How can you determine if something is a quaternary structure

• multiple subunits (different colors or N/C terminal indications)

Parallel vs antiparallel b-strands

parallel:

• need to loop back causing longer turns

• stable, but less stable than antiparallel

antiparallel:

• can have short loops since you can immediately flip back

• H bonds present between the backbone

• more stable due to optimal geometry for H-bond forming!

What amino acid is good in the hinge region of the b-turn and why

glycine: no R-group, highly flexible, low steric hindrance allowing proper folding

Proline: rigid ring structure increasing ability to make a sharp turn

What is the driving force to go from 2° → 3° structures

• r group interactions

• H-bond → stabilizing effect

hydrophobic regions + hydration layer

hydrophobic regions will interact and reduce the size of the hydration layer due to the reduced surface area of the nonpolar molecules

What helps stabilize the tertiary structure and how?

Van der waals interactions

a. Dipoles aligned with opposite charges close together

• permanent/temporary dipoles come in close proximity of another molecule causing an induced dipole

**When atoms are too close, there is strong repulsion. When atoms are farther apart, there is a weak attraction (at potential energy = 0)

*At lowest (dip) of potential energy, is the when there is optimal van der waals interactions

Disulfide bonds

stabilize the tertiary structure

• via oxidation (lose 2H+ and 2e-)

• reduction will use 2H+ and 2e-to separate the cysteines

Salt bridges

stabilize the tertiary structure

• has both electrostatic interactions AND hydrogen bonding

alpha-loop-alpha

alpha helices with a loop in the middle connected to another alpha helices

beta-turn or beta-hairpin

antiparallel b-strands connected by a b turn

beta-alpha-beta

parallel beta strands connected by alpha helix

greek key

4 antiparallel b-strands

• 1 → 2 → 3 (loops around to) → 4

Rossmamn fold

• alternating B-strands and alpha helices

• parallel b-strands

• alpha helices will allow the chain to conenct the b-strand while keeping them parallel

Key part:

alpha helix has a dipole, and since the helices are similarly arranged, their dipoles will be oriented in the same direction

• nucleotides are negatively charged due to the phosphate groups which will be attracted to the (+) dipole end of the alpha helix

• helps proteins bind with nucleotides

Four-helix bundle

just four alpha helices

TIM Barrel

Alternating alpha helices and parallel b-strands

• the arrangement is what will differ from the Rosman

• there are 7 beta strand residues in the actual length of the beta strand

Arrangement: folds into a barrel where the b-strands are inside, and the alpha-helices are outside

• this creates a cavity, and what binds inside the cavity will depend on the R-group lining it

Afinsens experiment + conclusion

Goal: try to determine where the instructions telling a protein how to fold are

Conclusion: amino acid sequence in teh primary structure contains the info necessary for the protein to reach a 3D structure

What he used in his experiment: RNase A, 8 cysteines (4 disulfide bonds), urea, and BME.

• Urea: disrupts non-covalent interactions (including hydrophobic effect) —> protein unfolds

• BME; reduces the disulfide bonds

Experiment one: First remove the BME then the Urea

• no BME = disulfide bond formation

• Urea present = unfolded proteins

• no folding means that the correct cysteines will not be brought near each other, and the cysteines may form incorrect/random disulfide bonds → you get a scrambled species

Scrambled species: due to the protein not being able to use all interactions encoded by the sequence due to the urea preventing normal folding

Experiment two: remove BME and urea at the same time

• normal folding interactions and correct disulfide bonds can form

• the protein will fold based on its amino acid sequence

  • the hydrophobic collapse and other noncovalent interactions can help get correct shape

• 100% native species forms

Impact of added a small amount of BME to the scrambled species after experiment one

• BME will reduce the incorrect disulfide bonds

• since there is now no urea, the normal noncovalent interactions can guide the protein towards its native shape and the correct cysteines will be brought to each other

Structural proteins

Cellular/organ/tissue support and structure

ex) tubulin, actin, collagen

Contractile proteins

muscle movement and cellular movement

ex) actin, myosin

Intermediate filaments

tension cable → resists change to cell

• long lives and resistant to mechanical stress

Microtubules (tubulin)

Movement (moving/pulling vesicles between organelles)

• cell division

• anchoring one cell to another

• cel shape + movement

ex) kinesin

Membrane ruffles + Lamellipodia

• structure of actin

• migration/movement: leading strand stretches, lagging strand contracts

  • lots of actin branching and presence at leading strands

Stress fibres

• structure of actin

• Resisting change and keeps the structure in the cell

• important for support + strength

Filopodia and cell spikes

• structure of actin

• antennas of the cell

  • determine if it is a good environment to move to and if we want to move there

Role of actin

• endo/phagocytosis

• anchoring to rigid structures of other cells via cell junction

What determines the structure of microfilaments

• structure and length

• determined by accessory proteins