Statistical Mechanics, Partition Function, Phase Transitions, Secondary Structure Formation

Biophysics 2018 Lecture 4, January 23
Statistical Mechanics,
Partition function, Phase
Transitions, Secondary
Structure Formation
Magnus Andersson
[email protected]
Recap from lecture 3
• Entropy in the hydrophobic effect
• Hydrophobic effect in protein folding
(hydrophobic collapse, molten globule)
• I.e. entropy critical in protein folding
• Boltzmann distribution
• Phase transitions introduced
• Partitioning of hydrocarbons
• Simple electrostatics
Today
• Foundations of statistical mechanics
• What do we mean by ‘stability’ or ‘instability’?
• The partition function

• Free energy & stable states
• Gradual changes & phase transitions
• Activation barriers & transition kinetics
• Helix and Sheet formation

Statistical Mechanics
Observations in a system
E is conserved
Consider all microstates of this system with energy E
# thermostat microstates Mtherm with the energy (E-ɛ)
Probability of observation is prop. to Mtherm(E-ɛ)
Define: Stherm(E-ɛ) = k*ln [Mtherm(E-ɛ)]
Just a unit constant
Entropy
⇥ ⇤
Stherm (E ✏) =  ln Mtherm (E ✏)
Now do series expansion; only 1st order✓matters - why?
◆
dStherm
Stherm (E ✏) = Stherm (E) ✏
dE E
Solve for M

Stherm (E ✏)
M (E ✏) = exp

 ⇢ 
Stherm (E) (dStherm /dE)|E
= exp ⇥ exp ✏
 
Observation of microstates
The probability of observing the small part in this state
is proportional to the number of microstates
corresponding to it
p / M (E ✏) / exp { ✏ [(dS/dE)|E /]}
1
(dS/dE)|E =
T
=k
Probabilities of states
probability of being in a state i
exp ( i /kB T )
wi (T ) =
Z(T )
Normalization factor
Z(T ) = exp ( i /kB T )

i
‘The partition function’
X
E(T ) = wi ✏i
i
X
S(T ) = wi Si
i
How do we calculate Si?

System distribution over
states
Consider N systems - how can we distribute them?
w1 w2 w3 wj
1 2 3 j
X
ni = wi N wi N = N
i
Question: how many ways can these systems 
be distributed over the j states?
Permutations
N!
= Stirling: n!≈(n/e)n
n1 !n2 !...nj !
n1 +...+nJ n1 nJ
= (N/e) / [(n1 /e) · · · (nJ /e) ]
= (N/n1 ) ...(N/nj )
n1 nj
= (1/w1 ) N w1
...(1/wj ) N wj
⇥ w1 w ⇤N
= 1/(w1 ...wj )
j
for N systems
w1 wj
= 1/(w1 ...wj ) for 1 system
...and the entropy becomes: X

S = kB ln M = kB wi ln(1/wi )
i
Free energy
X
E(T ) = wi ✏i
iX
S = kB ln M = kB wi ln(1/wi )
i
F (T ) = E(T ) T S(T )
X
= wj {✏j T [ kB ln wj ]}
j
If you know Z, you can calculate F exactly!
If you know Z approximately,
you can calculate F approximately!
System instability
System stability
Gradual changes
What does this correspond to? Examples?

Abrupt changes - all or none
ΔT = 4kBT*2 / (E2-E1)
When do things happen?
Free energy barriers
n# ≈ n exp(-∆F #/kBT) Ƭ(n/n#) ≈ Ƭ exp(∆F #/kBT)

⇥
t0 1 exp + F /kB T #
Transition rate: k0 1 = 1/t0 1

Can we apply this to
secondary structure?
• Alpha helix formation
• Beta sheet formation

Alpha helices
Hydrogen bonds: i to i+4
0-4, 1-5, 2-6
First hydrogen bond “locks” 

residues 1,2,3 in place
Second stabilizes 2,3,4 (etc.)

Alpha helix formation
• Hydrogen bonds: i to i+4
• 0-4, 1-5, 2-6
• First hydrogen bond “locks” 
residues 1,2,3 in place
• Second stabilizes 2,3,4 (etc.)

• N residues stabilized by N-2 hydrogen bonds!
Alpha helix free energy
• Free energy of helix vs. “coil” states:
H-bond free energy Entropy loss of fixating one
number of residues
residue in helix
F =F Fcoil = (n 2)fH-bond nT S
⇥
= 2fH-bond + n fH-bond TS
Helix initiation Helix elongation

cost cost
F = fINIT + nfEL
Alpha helix free energy
⇥ ⇥
exp ( F /kB T ) = exp fINIT /kB T exp nfEL /kB T
⇥⇤ ⇥⌅n
= exp fINIT /kB T exp fEL /kB T
= sn
⇥
s = exp fEL /kB T
⇥
= exp fINIT /kB T
⇥ ⇥
= exp fINIT /kB T = exp +2fH /kB T << 1
Equilibrium constant for helix of length n

How does a helix form?
First, consider ice in water - how large is water/ice interface?

n V r3
A r2 n2/3 Surface tension costly!
S = k ln(N)
Landau: Phases cannot co-exist in 3D
First order phase transitions means either state can be

stable, but not the mixture
Think ice/water - either freezing or melting 

3
n V r
2 2/3
A r n This does not happen in helix
But a helix-coil transition in a chain is 1D!
Interface helix/coil does not depend on n

ice/water: n molecules in ice, N in water

energy cost * n2/3 & entropy: k ln N
helix/coil: n residues in helix out of N in total
fINIT - kT ln (N-n) i.e. opposite to water/ice!
Helix/coil mixing
• Or: What helix length corresponds to the 
transition mid-point? fEL = fH T S = 0
• Assuming helix can start/end anywhere, 

there are N2/2 positions 
  S = k ln V k ln N 2 = 2k ln N
Fhelix ⇥ fINIT 2kT ln N
• At transition midpoint we have ΔF=0 & N=n0 
⇥
n0 = exp fINIT /2kT = 1/
Rate of Formation
Experimentally: Helices form in ~0.1μs! 
(20-30 residue segments)
One residue < 5 ns...
What is the  
limiting step?
Formation...
τ:1-residue  
• Rate of formation at position 1: 
⇥ elongation
 t
INIT0 = ⇥ exp fINIT /kT = ⇥ /
• Rate of formation anywhere (n0≈1/√σ): 

tINIT = ⇥ /
• Propagation to all residues: tn0 = ⇥ /
• Half time spent on initiation, half elongation!

Helix summary
Very fast formation
Both initiation & elongation matters
Low free energy barriers, ~1kcal/mol
Characteristic lengths 20-30 residues

Beta strands & sheets
How is this different 
from helices?
Interaction patterns?
Where are side 
chains pointing?
Can you think of 
differences for the 
folding/formation?
Beta sheet formation
• Experimentally: Can take hours to weeks!
• But sometimes just a millisecond. Why?
• Is it initiation- or elongation-limited?
• Beta sheet formation appears to be a typical

first-order phase transition!
Beta sheet formation
Hairpin
Beta sheet energies
• fβ: Free energy of residue inside a single beta hairpin,
relative to the random coil
• Δfβ: Extra edge free energy
• Total free energy at edge is fβ+ Δfβ
• U: Free energy of bend/coil per residue
• Since sheets can form we must have 

Δfβ>0 & U>0! 
Why?
Two Scenarios:
• fβ+ Δfβ<0: A single long beta hairpin 
will be more stable than coil. Only a single turn
required for formation
• fβ+ Δfβ>0: A single hairpin is less stable than coil.

Needs association with other residues into a beta
sheet. Activation barrier is the formation of a sheet
“nucleus”
Minimum strand length
• Consider the case when single hairpins 
are not stable. 
  F = U + 2N (f + f )
Turn All residues face an edge
• Association with a new strand maintains edge, and gives
us N new internal resides: 
F = Nf
• Formation of next turn: F” = U
• Minimum strand length: Nmin = U/( f )
Beta transition state
• Find the highest-free-energy intermediate: 
Single hairpin with a following turn
F # = U + 2Nmin (f + f ) + U = 2(U f )/( f )
The book goes into some 

detail to prove that this
is the lowest possible
transition state energy!
Why is that important?
Beta formation rates
• Initiation at a given point:
⇥
tINIT0 exp +F /kT #
• Initiation somewhere: tINIT0 /N
• Initiation is entirely time-limiting

⇥
• Total formation time: t exp F /kT /N
#
• And remember that we had: 

F # = U + 2Nmin (f + f ) + U = 2(U f )/( f )
Beta formation rates
• Rate depends on β-structure stability: 
t ⇥ exp [A/( f )]
• Exponential dependence  
on residue beta stability  
explains wide range of  
formation times  
observed in experiments!
Beta sheet summary
Unstable sheets are extremely slow 
to form (hours to weeks)
Stable sheets can form in milliseconds
Significant free energy barrier
Beta sheet folding is a first-order 

phase transition
Helices vs. sheets
Helix
Local h-bonds
Gradual (but fast) growth
Low initiation barrier
Sheets
Non-local h-bonds
Collective interactions; all-or-nothing
High initiation barrier - very slow formation
Misfolding - Prions
Misfolded form (right)

is protease resistant
The in vivo state is only the
second best here - but the
free energy barrier to the
lowest is very high!
Study questions
1. Understand E, H, S, F, G - what do they represent?
2. What is the partition function?
3. Explain stability vs. instability.
4. What characterises a phase transition?
5. How is free energy barriers related to reaction rates?
6. What is the hydrogen bond pattern in the most common helix?
7. What free energy components do you think (a) favor (b) disfavor helix folding?
8. What free energy components do you think (a) favor (b) disfavor sheet folding?
9. Why do you get a helix dipole?
10. What is helix capping?
11. Why is sheet formation a phase transition while helix formation is not?
12. Why is the range of formation rates for sheets large while it is not so for helices?
13. Explain the difference between macrostates and microstates!
14. Based on what you now know, how would you explain entropy?
Hand-in tasks 1 & 2
• Hand-in task 1 
- Enthalpy & Entropy 
- Boltzmann distribution
• Hand-in task 2 
- Secondary structural stability and formation 
(Lecture 4 slides & Finkelstein p. 103-114)
• All information & programs on Bilda
• Deadline Hand-in task 1: Feb 15
• Deadline Hand-in task 1: March 2

Covered material and
suggested reading
• Nordlund  
- Chapter 14 (but better described in Finkelstein)
• Finkelstein 
- Chapter 7,8,9
For lecture 5
• Looking at real proteins
• Complex assemblies from simple 
α-helix/β-sheet building blocks
• Supersecondary structure & “motifs”
• Fibrous proteins
• Globular proteins
• α-helix, β-sheet proteins
• Mixed structures

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Biophysics 2018 Lecture 4, January 23

• What do we mean by ‘stability’ or ‘instability’?

• The partition function

• Gradual changes & phase transitions

• Activation barriers & transition kinetics

• Helix and Sheet formation

p / M (E ✏) / exp { ✏ [(dS/dE)|E /]}

Z(T ) = exp ( i /kB T )

How do we calculate Si?

...and the entropy becomes: X

What does this correspond to? Examples?

n# ≈ n exp(-∆F #/kBT) Ƭ(n/n#) ≈ Ƭ exp(∆F #/kBT)

Transition rate: k0 1 = 1/t0 1

• Beta sheet formation

Hydrogen bonds: i to i+4

0-4, 1-5, 2-6

First hydrogen bond “locks”

Second stabilizes 2,3,4 (etc.)

• Second stabilizes 2,3,4 (etc.)

Helix initiation Helix elongation

Equilibrium constant for helix of length n

First, consider ice in water - how large is water/ice interface?

First order phase transitions means either state can be

Think ice/water - either freezing or melting

Interface helix/coil does not depend on n

ice/water: n molecules in ice, N in water

• Assuming helix can start/end anywhere,

One residue < 5 ns...

• Rate of formation anywhere (n0≈1/√σ):

• Half time spent on initiation, half elongation!

Very fast formation

Both initiation & elongation matters

Low free energy barriers, ~1kcal/mol

Characteristic lengths 20-30 residues

• Experimentally: Can take hours to weeks!

• But sometimes just a millisecond. Why?

• Beta sheet formation appears to be a typical

• Δfβ: Extra edge free energy

• Total free energy at edge is fβ+ Δfβ

• U: Free energy of bend/coil per residue

• Since sheets can form we must have

• fβ+ Δfβ>0: A single hairpin is less stable than coil.

The book goes into some

• Initiation somewhere: tINIT0 /N

• Initiation is entirely time-limiting

• And remember that we had:

Stable sheets can form in milliseconds

Significant free energy barrier

Beta sheet folding is a first-order

Misfolded form (right)

• All information & programs on Bilda

• Deadline Hand-in task 1: Feb 15

• Deadline Hand-in task 1: March 2

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First hydrogen bond “locks” 

Think ice/water - either freezing or melting 

• Assuming helix can start/end anywhere, 

• Rate of formation anywhere (n0≈1/√σ): 

• Since sheets can form we must have 

The book goes into some 

• And remember that we had: 

Beta sheet folding is a first-order 