An introduction to proteins:

Enzymes:
Kim et al., Science 2017, 355 (6322), eaag2355.
Mehrabi et al., J Am Chem Soc 2019, 141 (29), 11540.
GPCRs:
Nobel Prize Lecture on GPCRs 2012
Nobel prize Lecture on GPCRs (II) 2012
MPro 1
GPCR Database on everything Zhang et al., Science 2020, 368 (6489), 409.
I. An introduction to proteins and protein folding
II. An introduction to enzymes.
III. An introduction to GPCRs

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In this module, we’ll make use of the the Protein Data Bank (PDB) which is a free online depository of ~210,000 known protein
structures & other important biological macromolecules and over 106 predicted structures. If you are new to protein structure,
function, and common visual representations of proteins, the Protein Data Bank website has a separate tab (PDB101) with useful
instructional links & videos.

Protein structure & function - Introductory video

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Identify the key functions associated
with proteins

4
What is this amino acid? Is it
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hydrophobic or hydrophilic?
What might be the enthalpic and entropic
consequences to dissolution in water?

What would happen if we added each of these amino

acids to a beaker consisting of 50% octanol and 50%
water?

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Formation of polypeptide by a condensation reaction:
If we combine any two amino acids in water will they
eventually become a dipeptide?

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8
What drives protein folding?

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How enzymes work

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Association of 9
enzymes powers
ATP generation
through
glycolysis and
TCA cycle

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Enzymes catalyze nearly every biological
process associated with synthesis, cell
renewal and growth

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Specific amino acids readily act to activate waters,
donate protons, abstract protons, or participate in
the active site

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Another great interactive composition of protein structures,
exemplifying specific functions can be found here:
Protein Map of Structures & Function

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II. Activation
III. An introduction to GPCRs

G protein-coupled receptors (GPCRs) are

cell-surface receptors mediating the
responses of 2/3rds of human hormones
and 1/3rd of drugs. Each GPCR can bind
several transducers: G proteins, GPCR
kinases (GRKs) and arrestins leading to
distinct intracellular signaling networks
and functional outcomes

GPCRs often exhibit pronounced

enhancement of specific signalling
pathways but must do so without
expending chemical energy (e.g. ATP
hydrolysis and coupling).
1. How can they overcome these energy
barriers?

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Family A GPCRs consist of a host of well-known motifs or micro-switches involved in activation – DRY motif, NPXXY,
hydrophobic belt, toggle switch, and the water-Na+ network

Vickery et al Structure (2018) 26, 171–180 16

Na+ binding sites are highly conserved in Family A GPCRs

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In nanodiscs, 23Na NMR reveals a “long lived” strongly bound state in addition to one or
more weakly bound sodium ions exchanging with bulk solvent on a millisecond timescale.
1
2↔3

23
A2A HDL H2O Na Shift
No Cholesterol 0.265
23Na T1 filter

0.260

Shift, ppm
0.255 1
kd=13.6 ± .8 mM
secondary binding site
0.250
2
0.245
0 20 40 60
NaCl, mM

3
18
Pichugin et al, (unpublished)
40-125 mM [Na] gives rise to enhanced Hill Slope & IC50, even though Na+ is recognized as a NAM
Pichugin et al, (unpublished)

NECA_vs_NaCl 50 mM NaCl, IC50 = 42.5 +/- 0.5 nM, Hill Slope = 2.24 +/- 0.05

500
3mM Summary table: Nonlin fit of NECA_vs_NaCl
GTPAse % increase

400 6.25mM 2.5 150

IC50
12.5mM 2.0 HillSlope
300
25mM 100

Hill Slope

IC50, nM
1.5
200 50mM
1.0
125mM 50
100
250mM 0.5

0 500mM 0.0 0
1 10 100 1000
0
10

00
1

00

0
0
1

00

00
0.

10

00
10

NaCl, mM
10

0
10

10

NECA, nM 125 mM NaCl, IC50 = 31.9 +/- 1 nM, Hill Slope = 1.82 +/- 0.1

We postulate Na switches from a primary to a secondary binding site, thereby switching from stabilizing an inactive state to an
active G-protein coupled state and enabling signalling!

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Lessons from these videos:

* Protein Function: defense (e.g. antibodies), signaling, enzymes, transport, storage (e.g. ferritin), structure
* Enzymes catalyze chemical reaction rates by as much as 1011 or more and by stabilizing the transition state, they lower the
activation barrier so that the reaction is energetically accessible.
* Metals and/or organic cofactors are a common facet of enzymes (e.g. iron-sulfur complexes in cis aconitase)

2. What do all 21 amino acids have in common?

3. How are alpha-helices and beta-sheets characterized?
4. What drives protein folding?
5. What is a hydrogen bond?

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 Forces stabilizing proteins
FEBS Letters 588 (2014) 2177–2184
See https://www.youtube.com/watch?v=yZ2aY5lxEGE to understand how we visualize proteins
(1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2
group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability.

(2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term
that depends on the removal of the side chains from water and, more importantly, the
enhanced London dispersion forces that result from the tight packing in the protein interior.

(3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond
on folding contributes 1.1 ± 0.8 kcal/mol to protein stability.
(4) The contribution of hydrogen bonds to protein stability is strongly context dependent.
(5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein
stability.
(6) Polar group burial can make a favorable contribution to protein stability even if the
polar group is not hydrogen bonded.
(7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein
stability.
What’s the dissociation enthalpy of an sp3 CH bond? 337 kJ/mol
What is thermal energy? 2.4 kJ/mol

6. Why is thermal energy relevant to protein folding kinetics? 21

 Protein interiors consist of lots of tightly packed non-polar side chains

On average, proteins bury 85% of their non-polar side chains and form 1.1 hydrogen bonds per residue when the protein folds

Greatest possible packing density achieved from a lattice of spheres: 74%

7. How can a protein pack better than close-packed spheres?

8. What is the consequence of such close packing in the protein interior to van der Waals forces?

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An FCC lattice, wikipedia
9. Why would London Dispersion forces be even higher in the protein interior?

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Interior hydrogen bonds contribute 1.1 ± 0.8 kcal/mol to protein stability
This is done by examining the change in the free energy of unfolding resulting from mutations (i.e. by removing a single
hydrogen bond in a protein and examining the resultant decrease in folding free energy stability)

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Hydrogen bonding in proteins and fold stability

10. Why do buried polar groups often give rise to increased protein stability?

11. Hydrogen bonds can be more than 1 kcal/mol stronger in a hydrophobic environment. Why?

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Disulfide bonds greatly increase protein fold stability but predictably decrease configurational entropy

Based on experimental evidence, the contribution of a disulfide bond to stability can often be estimated reasonably well with
this equation
ΔS = -2.1 –(3/2) R ln (n)
where n is the number of residues in the loop formed by the crosslink and R is the gas constant. ΔS refers to the decrease in
conformational entropy associated with the disulfide. The equation predicts that the stability will be increased by 3, 4, and 5
kcal/mol by loops of 15, 45, and 135 residues, respectively.

Interior Salt links

Ion pairs on the surface generally contribute less than 1 kcal/mol to the stability. However, a buried salt bridge can contribute
more than 4 kcal/mol to the stability, but the number of buried salt bridges in proteins is small so they do not make a large
contribution to stability.

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Conclusions:

“The experimental results from many groups over the past few decades have confirmed that hydrophobic interactions do make
the major contribution to protein stability but that hydrogen bonds also make a large contribution. In addition, global analyses
based of the experimental results support this and show that hydrogen bonds make a favorable contribution to protein stability.
Applying the results presented in this review to 22 proteins containing from 36 to 548 amino acids … on average, hydrophobic
interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability

12. Can we use evolutionary data to help us understand protein folding?

See Video

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Review
(1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2
group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. How is this evaluated?

(2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term
that depends on the removal of the side chains from water and, more importantly, the
enhanced London dispersion forces that result from the tight packing in the protein interior.
How well packed the interior of a folded protein?
(3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond
on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. Why are hydrogen bonds so
variable in free energy?
(4) The contribution of hydrogen bonds to protein stability is strongly context dependent.
(5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein
stability. (Statistically it is roughly 60% van der Waals and 40% hydrogen bonds)
(6) Polar group burial can make a favorable contribution to protein stability even if the
polar group is not hydrogen bonded. How?
(7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein
stability.

What’s the dissociation enthalpy of an sp3 CH bond? 337 kJ/mol

What is thermal energy? 2.4 kJ/mol 28