24OCT2022

• Peptide bond = condensation reaction

• Planar = because of thermodynamics ( steric hindrance )

• Rigid = double bonds ( pi bonding )

  • Because of Resonance structure , 40% of it is in a double bond

• Because peptides are planar and rigid , it gives us our “torsional angles” aka $\psi$$\psi$ and $\varphi$$\phi$

• $\varphi$$\phi$ = bond between the Nitrogen and the alpha carbon

• $\psi$$\psi$ = bond between alpha carbon and the carbonyl carbon

• Both can rotate freely from -180 to +180

  • In reality they don’t get this full range because of steric hindrance

Determining Amino Acid Sequence

• Edman = chemical purification

• Mass spec = “more modern”

• Determining the amino acid content gives you guide-rails for where you are , orientation , gives you molar ratios

  • Weak Acid hydrolysis = break N-terminus peptide bond
  • Strong acids can break apart the entire peptide and all its bonds

• Finding the amino terminus tells you how many subunits you have

• Add a chemical that reacts with the N-terminal amino group

• Then you subject it to strong acid , and break it apart into free amino acids

  • One of them still has the label

    • You then purify

• Then you repeat with labeling the same amino terminus , but you add a mild acid , and cleave only the N-terminus

  • Then purify , that tells you what is in position one

• Then repeat with new N-terminus labels , and then break with weak acid

  • Purify , record latest position

• You can only do Edman for a short peptide

  • Errors propagate with each cycle

• To synthesize longer than 100 peptide length

  • Use two separate experiments on original peptide ( chymotrypsin ) and trypsin

  • These cut at different spots

    • Now you can overlap the fragments and see where they match

  • Determining which proteases to use is crucial

    • You want to minimize the number of cuts , makes it easier to find overlap matches

• ESI and MALDI = soft ionization technique , doesn’t blow the peptide to smithereens , breaks it into chucks

• ESI :

  • can be combined with HPLC

    • Separates out protein first , then identify with HPLC

  • MALDI :

    • has a much higher throughput

      • Adding a salt solution makes it form a crystal

    • Then you can shoot a layer at the crystal , causes ionization

    • MALDI is even “softer” than ESI

  • MS-MS :

    • Successive iterations of MS experiments

• Problem :

  • Tyrosine-

  • Leucine-

  • Glycine-Phenylalanine-Glycine

  • First bullet point = tells us molar ratio only

  • 2nd bullet point = tells us peptide was labeled on N-terminus , shot it apart with an acid , tells us that the N-terminus was tyrosine , also that there is only 1 tyrosine

  • 3rd bullet point = the only way chymotrypsin could be applied and it cleaves a peptide length of 3 , is for phenylalanine to be on the end

    • Gly-Gly-Phe

  • We know leucine is “free”

    • Via trial and error , we know where chymotrypsin cleaved ,

  • Structure must be :

    • Tyrosine-Glycine-Glycine-Phenylalanine-Leucine

  • If there is commas or the ( 2 ) it is not ordered to N - C , it just tells you similar to the ratio

For question 3 :

• 0.4 M becomes

  • 4.0 = 3.75 + log ( ( 0.2 + x ) / ( 0.2 - X ) )

• Use X with concentration of sodium hydroxide to get the liters6

Secondary Structure

• $\mathrm{\Delta }S$$\Delta S$ of water = favorable

• $\mathrm{\Delta }S$$\Delta S$ of protein is unfavorable

• $\mathrm{\Delta }S$$\Delta S$ of protein folding = unfavorable

• The majority of what makes protein folding have a negative delta G comes from the enthalpy term

• Hydrophobic effect = what jump starts protein folding

  • The driving force of protein folding
  • The hydrophobic regions on the protein are super uncomfortable
  • The polar regions still have the ability to hydrogen bond with water
  • We still wan’t to get the hydrophobic areas “together”
  • $\mathrm{\Delta }{S}_{folding}=\mathrm{\Delta }{S}_{solvant}+\mathrm{\Delta }{S}_{protein}$$\Delta S_{folding} = \Delta S_{solvant} + \Delta S_{protein}$
  • + = (-) + ( + )

• The peptide backbone dictates hydrogen bonding , NOT the R groups

$\alpha$$\alpha$-Helix

• simple
• Maximizes the hydrogen bonding between amino acids
• Around 3.6 Amino Acids / 1 Turn
• Can interact with membranes
• Distance between rings = 5.4 angstroms
• 40% of protein structure is alpha-helical
• Limited proline = makes it too rigid , proline-kinks
• Limited glycine = glycine is extremely flexible , makes it hard to form helix
• Proline and Glycine are really good for forming the turns in beta sheets

$\beta$$\beta$-Sheets

• backbone is in a zig-zag conformation

• Multiple layers combine via hydrogen bonding

• Layers can be parallel or anti-parallel

• Parallel :

  • Hydrogen bonds are strained off their axis
  • Not as thermodynamically stable

• Anti-Parallel :

  • Hydrogen bonds are directly off their axis , not strained
  • More thermodynamically stable

• Number of hydrogen bonds is dictated by the number of amino acids in the backbone

• The favorability of hydrogen bonds is dictated from it being either parallel or anti-parallel

Which of the following peptides would more likely to form an alpha helix ?

• The first one , no glycine and proline
• How many turns would it have ?
• 15 / 3.6 = ??

Higher Order Structure of Proteins

• tertiary structure is stabilize by weak interactions

• Fiberous = scaffolding , and give structure

  • Collagen , keratin
  • Make up the majority of the “volume”

• Globular = cytosolic protein

  • Enzymes
  • We have more globular proteins , majority of the “population”

Keratin

• nails and hair

• Rich in hydrophobic amino acids

• 2 stranded , twisted together

  • Each strand is a right-handed helix
  • The two strands winding around each other aid in its growth in “length” = more stability

• Stabilized with di-sulfide bonds

• Naturally curly hair = di-sulfide bonds

Collagen

• skin , joints , bone

• Connective tissue

• Triple helix

• NOT an $\alpha$$\alpha$ helix

• Really rich in proline and glycine

  • Proline = rigid
  • Glycine = flexible

• The helix is much more tightly wrapped

  • Around 3 amino acids per turn

• G-X-Y pattern

• Different types of collagen mixed and matched produces different structures

• Fibers need to be cross-linked

  • Osteogenesis imperfectis ( brittle bone disease )

Silk Fibroin

• Anti-parallel beta sheets

  • Very rigid in the plane

Globular Proteins

• There are regions that automatically fold together

  • Primary sequence dictates secondary and tertiary sequence

• If you assume a 100 amino acid protein :

  • 10 confirmations / amino acid

  • also assume it takes ${10}^{-13}$$10^{-13}$ seconds to sample each confirmation

    • It would take ${10}^{77}$$10^{77}$ years to fold 1 protein

• Intrinsically the protein is skipping a lot of the sampling folding steps

  • The primary sequence just dictates forced folding angles , and these happen automatically

Motifs

• common structural elements when secondary structures come together

• Parallel needs to be joined by a longer segment

  • Beta-Alpha-Beta motifs allows for these longer segments to form

Quaternary Structure

• most abundant motif / subunits = $\alpha$$\alpha$
• If there is a tie , give preference to the higher molecular weight
• Fetal hemoglobin has a higher affinity to oxygen , similar to adaptation in high altitude

Misc

• NMR and X-Ray can be used to identify static structure

• EPR = good for looking at dynamics / movement

  • Put 2 labels on protein , the closer their proximity = greater the signal

• ESI and MALDI = soft ionization techniques

• Organic Mass Spec = blowing apart the molecule

Problem ?

• How would you classify this protein ?

• Has both alpha and beta

• Are they segmented ? All the alphas together , and all the betas together ?

  • They look like they are separated

• $\alpha$$\alpha$ + $\beta$$\beta$ , the “plus” is because they are separated

Domains

• can be separated and still perform its original functions
• Large , Independent areas of the protein that have individual functions

Protein Dynamics

• organic solvents disrupt hydrophobic effect

• Proteins have a very steep melting curve

  • Sigmoid curve = binary on or off

    • Once it starts folding , the rest will go really quickly

  • All-or-none event essentially

• Leventhall’s Paradox :

  • Available number of confirmations is too large to find the perfect one randomly
  • It must be able to rule out huge areas of confirmation that it will never go into
  • Figure 4-27 , 6th edition
  • When disulfide bonds reform , it gets scrambled as they form new bonds “randomly”

Figure 6-38 : Ribonuclease Folding Experiment

• Proteins when they go to reassemble , don’t necessarily reassemble perfectly.

  • Some proteins use different chaperone proteins to help them fold as they leave the ribosome

• For instance , the C-motif on insulin acts as a chaperone for the correct form of the active insulin

• “Pro-version” of insulin gets cleaved off to create active version of insulin

Enthalpic or Entropic Factors

• Asdf

Folding Intermediates

• Sometimes you need to break co-valent or non-covalent interactions to proceed to the next folding intermediate

  • This needs to be energetically favorable
  • Size of the “hump” = longer it takes to fold the next intermediate
  • For each attempt to fold to the next intermediate , requires more and more time
  • Multiple activation energies for each transition state , the more transition states / folding intermediates , the longer it takes to fold
  • Also , the more stable the intermediates , the longer it takes to fold

• Certain segments of the protein fold first , the rest folds around it

• Protein-Folding-Accessory-Proteins aid in folding

Chaperones

• asdf

Problem : Why does the Gro EL/ES system only function in one direction?

• the inside cavity provides a hydrophobic environment

• the ATP hydrolysis used to power , creates an energy barrier almost impossible to reverse

  • Once you hydrolyze ATP , the Delta G to reattach phosphate group is almost impossible

Myoglobin and Ligand Binding

• $K_d\approx P_{50}$$K_d \approx P_{50}$ = same concept , just for gas

• $K_d$$K_d$ = normal concentration units

• $P_{50}$$P_{50}$ = gas partial pressure

• $K_D\frac{1}{\propto }$$K_D\ \frac{1}{\propto}$ affinity

• $P_{50}\frac{1}{\propto }$$P_{50}\ \frac{1}{\propto}$ affinity

• Number of binding sites occupied = “normalization” , because hemoglobin has 4 binding sites , and myoglobin only has 1 binding site

• $K_d$$K_d$ = concentration it takes to reach “half saturation”

  • If it takes more concentration to reach half saturation , its higher affinity ???

Myoglobin and Hemoglobin

• Histamine attaches myoglobin to the Iron co-factor

• Proximal Histidine = directly attached to iron

  • Part of the “F helix”
  • Distal Histidine = attached via electrostatic interactions to further stabilization

• Iron in the center , porforin ring surrounds it

Structure of Myoglobin

• Proximal vs Distal Histidine
• Myoglobin has low P50 = good at binding oxygen
• Myoglobin has to high of a binding affinity to oxygen for it to work as a transport protein

Hemoglobin

• as you add oxygen to hemoglobin , the affinity increases as you add more oxygens

Problem :

• a protein binds a ligand …..

O₂ Transport

• shifting the partial pressure to the right is not enough

  • We need some conformational change

• It starts in the low affinity state

  • Once it reaches a certain partial pressure of oxygen , it changes from low affinity state to high affinity

    • Becomes saturated more quickly now ( cooperativity ) ( positive homotrophic allosteric effector )

• When first oxygen binds , it moves heme to be in plane with the iron

  • Normally , there is an amino acid that causes the Tense / Strain state

  • That causes the proximal histidine to move with the iron

    • The proximal histidine is attached to the F-helix

      • This moves the F-helix

        • causes 15-degree rotation at interface between alpha and beta subunits ?

• Deoxystate = 4 oxygens are closed off

  • Once 15 degree rotation happens , it moves to high affinity state

    • Oxygen binding sites are more open and accessible

pH Effect on O₂ Binding to Hemoglobin

• Hemoglobin in the T-state has a few hydrogens that can be protonated

• When oxygen is added , hemoglobin binds to oxygen and releases protons

  • If you added acid , you are pushing the equilibrium to the left

    • Shifting from R-state to T-state

      • Shifts the partial pressure of oxygen / binding affinity graph to the right

        • This increases the $\mathrm{\Delta }y{O}_2$$\Delta y\ O_2$

T-state = left hand side

R-state = right hand side

• Lactic acid lowers the pH , causes left shift

  • Makes it easier for oxygen delivery ?

• Hydrogen on hemoglobin deprotonates

  • PKa values can change

• The protein would be “inside-out” with hydrophobic regions on outside , hydrophobic on inside

pH Effect on $O_2$$O_2$ Binding to Hemoglobin

• T state = Hydrogen bound to hemoglobin
• R state = Hydrogen unbound to hemoglobin

2,3-BPG

• In the R-state , the positive charges can no longer interact with 2,3-BPG , causing 2,3-BPG to leave

• 2,3-BPG = negative allosteric effector , causes right shift , increases delta y $O_2$$O_2$

• Adult hemoglobin has positively charged histidine

  • Binds 2,3-BPG

• Fetal hemoglobin has the histidine swapped out for serine

  • Does not bind 2,3-BPG

    • Left Shift

• Oxygen = positive homotrophic effector
• PH = negative heterotrophic
• 2,3-BPG = negative heterotrophic

Misc

• It takes longer to breathe in $O_2$$O_2$ than it does to expel $C{O}_2$$CO_2$

• Hyperventilation = left shift = decrease delta-y-O-2

• Hemoglobin without allosteric effectors has huge left shift , very ineffective for transport

  • adding the effectors causes right shift , increases delta-y-O-2

    • Effective for transport

Sickle Cell

• When hemoglobin is in R state , the glutamate is not exposed
• When it deoxygenates ,
• In Sickle cell , see video transcription
• Fetal hemoglobin can work as a blocker to prevent polymerization during sickle cell anemia

Problem

• Excess 2,3-BPG is bad for sickle cell anemia ?

  • Keeps it in the T-state longer ,

    • Increases amount of potential interactions with the valines that stick out , forming polymers

• Body is trying to compensate , but it doesn’t know it will cause the formation of polymerization

• Alpha Helixes tend to form with each other