!"#$%&'() "( +"','-.

/0. 10%$-2 ") "34'$#%(# "( 5"','-.6

180
o
!
!
90
o
Morikis, Roy, Newlon, Scott, Jennings (2002) European Journal of Biochemistry.
Hydrophobicity vs charges
7,21#$')#%&1 1%,18,%&'() 8)"(- 1'(&(883 9"2,21#$"1 3'92,) :'$ 4$'#2"()
; 4$'#2"( )8$$'8(929 5. <%#2$ 1$2%#2) 9"2,21#$"1 5'8(9%$"2)
!02 2,21#$')#%&1 4'#2(&%, -2(2$%#29 5. % 4$'#2"(
The activation helix-coil transition
Morikis, Elcock, Jennings, McCammon (2003) Biophysical Chemistry
His108
His132
His137
His119
His121
Catalytic site
His108
His132
His137
His119
His121
Catalytic site
GART Helix-coil transition & catalysis
NSP
In association with
BioMed Central
2004 New Science Press Ltd new-science-press.com

Catalytic triad. The catalytic triad of aspartic acid, histidine and serine in (a)
subtilisin, a bacterial serine protease, and (b) chymotrypsin, a mammalian
serine protease. The two protein structures are quite different, and the elements
of the catalytic triad are in different positions in the primary sequence, but the
active-site arrangement of the aspartic acid, histidine and serine is similar.
Functional motif
Identifying motifs from sequence alone is not straightforward
His40 Ser195
Asp194
Ile16
Profactor
His40
Ser195
Asp194
Ile16
Zymogen
His40
Ser195
Asp194
Ile16
Ile16
Profactor
Zymogen
&
His57
Ser195
Asp102
Profactor
His57
Ser195
Asp102
Zymogen
His57
Ser195
Asp102
Profactor
Zymogen
&
Profactor
Arg218
Asp189
Ser195
Zymogen
Arg218
Asp189
Ser195
Profactor
Zymogen
&
Arg218
Asp189
Ser195
Arg218
Profactor Zymogen
1HFD 1FDPa
pK(app) pK(app)
Ile16 7.5 7.3 7.4
His40 6.3 2.5 2.8
His57 6.3 8.6 8.0
Asp102 4.0 -0.7 1.5
Asp189 4.0 0.2 2.7
Asp194 4.0 -2.0 -0.6
Arg218 12.0 14.8 14.2
Res. No. pK(model)
Factor D: Interactions between residues with unusual pK
a
values
Backbone acid-base equilibrium for free amino acids
NH
3
+
COO
-
NH
2
COOH
Histidine acid-base equilibrium (backbone & side chain)
backbone Side chain backbone
Histidine tautomers
Rare
neutral charged neutral
pK
a
values for free amino acids in solution
Acidic residues:
C-ter: 3.8
Asp: 4.0
Glu: 4.4
Cys: 8.3 (non S-S bonded)
Tyr: 9.6
Basic residues:
His: 6.3
N-ter: 7.5
Lys: 10.4
Arg: 12.0
K
a
AH A

+ H
+
RT
ion
G
e
!
"
=
+ "
=
[AH]
] ][H [A
a
K
[AH]
] [A
log
a
pK pH
!
+ =
a
logK
a
pK ! =
Henderson-Hasselbalch
] log[H pH
+
! =
Side Chains
0 2 4 6 8 10 12 14
-12
-10
-8
-6
-4
-2
0
2
4



25mM
50mM
75mM
100mM
125mM
150mM
0 2 4 6 8 10 12 14
-60
-40
-20
0
20
40
60


Q(C3d-CR2)
Q(C3d)
Q(CR2)
!Q
pH and ionic strength effect on association
C-ter
Asp
Glu
His
N-ter
Tyr
Lys
Arg
C
h
a
r
g
e

"
"
G
i
o
n

(
k
c
a
l
/
m
o
l
)

pH
Isoelectric points
50 mM
"<Q>=0
) Q Q Q ( RT .
pH
) pH ( G
CR d C CR : d C
assoc
2 3 2 3
303 2 ! " # ! " # ! "
=
$
% $
Morikis & Zhang (2006) J Non-Cryst Solids
Tanford, 1970
Free proteins in
random diffusion
Encounter
complex
Final
complex
Recognition
Long-range
interactions:
electrostatic
macrodipoles
Binding
Long-range electrostatic interactions
Short-range interactions:
Electrostatic, hydrophobic,
H-bond, van der Waals
specific residues
Removal of H
2
O from interface
Local structural rearrangements
Association = Recognition + Binding
+ :
Association
"G
1,ion
(pH) "G
2,ion
(pH)
"G
complex,ion
(pH)
"G(neutral)
"G(pH)

+
+

+

+

+

+

+

+

+
+

+

+
+

+

+

+
Neutral
Ionized
Ionization
process

+

+

+

+
+

+

+
+


0 2 4 6 8 10 12 14
-60
-40
-20
0
20
40
60


Q(C3d-CR2)
Q(C3d)
Q(CR2)
!Q
C
h
a
r
g
e
pH
0 2 4 6 8 10 12 14
-60
-40
-20
0
20
40
60


Q(C3d-CR2)
Q(C3d)
Q(CR2)
!Q
C
h
a
r
g
e
pH
0 2 4 6 8 10 12 14
-12
-8
-4
0
4
8
12



25mM
50mM
75mM
100mM
125mM
150mM
!
!
G
i
o
n
(
k
c
a
l
/
m
o
l
)
pH
0 2 4 6 8 10 12 14
-12
-8
-4
0
4
8
12



25mM
50mM
75mM
100mM
125mM
150mM
!
!
G
i
o
n
(
k
c
a
l
/
m
o
l
)
pH
Morikis &
Zhang (2006)
J Non-Cryst
Solids
u
f
i
o
n
G
!
"
"
0.1 M
1 M
2 3 4 5 6 7
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5


AMS
D7N
D7K
2 3 4 5 6 7
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0


AMS
D7N
D7K
pH pH
D3
E38
E47
E9
D23
D7
D67
D80
R78
R33 K54
K86
R6
K63
R93
R30
Improvement of
Fibronectin stability:
Scaffold for
Monobody design
Mallik, Zhang, Koide, Morikis
(2008) Biotechnology Progress
u f
neutral
G
!
"
u f
G
!
"
f
ion
G "
u
ion
G "
u f
neutral
G
!
"
u f
G
!
"
f
ion
G "
u
ion
G "

+

+

+
+

+
+
+

!G (neutral)
h-c
!G (pH)
h-c
!G (pH)
h,ion
!G (pH)
c,ion
h: helix c: coil
neutral
ionized
Folded ! Unfolded Helix ! Coil
pH dependence of conformational transitions
Isoelectric focusing

Isoelectric point (pI): pH at which the biomolecule (or ampholyte)
has zero charge

It is used to separate biomolecules

Suppose that we can create, in a gel or capillary, a stable pH
gradient between the anode and cathode

Molecules would migrate until each reached the pH equaled its pI

Bands would form at different points in the pH gradient

Bands would remain sharp, as if a biomolecule diffused away from
its pI position it would experience a force pulling it back
Graphical demonstration
How such a pH gradient can be established and maintained?

The cathode and anode departments become basic and acidic,
respectively through the reactions
2 2
2
4 4 2
2 2
O e H O H
H e H
+ + !
! +
" +
" +
Electrolysis of water

The pH changes resulting from these reactions are confined to
regions near the electrodes

Ampholytes with lower pI concentrate near the anode, whereas
those with higher pI concentrate near the cathode
Example
Example
OFarrell
technique
(2D)
Charge
Molecular
weight
Proteomics experiment: MS fingerprinting
2D gel
electrophoresis:
separation
according to
charge (pH) &
molecular weight
Properties of proteolytic enzymes
Strategy for determining protein sequence from proteolytic
fragments
Peptide sequencing by
tandem MS spectrometry
(MS/MS)
CID:
collision-induced dissociation
In a chamber filled with a
neutral gas (Ar or Xe) that
breaks the peptide backbone
Tandem MS: coupling of
two or more experiments
TOF: Time-of-flight mass spectrometer
Positive ions
of the
substance to
be analyzed
2 1
2
2
2
1
/
m
ZeES
v
ZeES mv
!
"
#
$
%
&
=
=
2
2 1
2
2
!
"
#
$
%
&
=
!
"
#
$
%
&
= =
D
t
eES
Z
m
D
ZeES
m
v
D
t
/
Field region
Ion generation
Units: Daltons
1 Da = 1 g/mol
Mass spectrometry (MS) of biomolecules

Determination of biomolecular mass

Determination of biomolecular sequence

Protein identification (proteomics)

Study of conformational transitions, protein folding, and protein
interactions in combination with H/D exchange
Mass spectrometry (MS) of biomolecules

Measures degradation products resulting from their collisions with
electrons ! molecular ions

Does not actually measure mass but m/Z (mass over charge) ratio

Molecular ions with smaller masses or larger # of charges have
higher velocity, which is used to resolve various molecular species
according to m/Z
The molecule of interest as a charged ion is placed into the gas
phase, then the molecular species must be separated according
to their masses, and finally the molecular ions must be detected
Difficulty in ionizing proteins compared to small molecules

For small molecules: we simply heat the sample

For proteins: MALDI or ESI

MALDI: matrix-assisted laser desorption/ionization
! Molecular mass measurement
! Study of proteins up to 300,000 molecular mass

ESI: electrospray ionization
! Molecular mass & sequence determination
! Study of proteins up to 500,000 molecular mass
Very small amounts of sample
! ESI MS: femtomole-picomole
! NanoESI MS: zeptomole-femtomole
! MALDI MS: femtomole
Matrix absorbs strongly at the laser wavelength
MALDI MS
Example of MALDI spectrum

Molecular mass measurement
More than one ionic species are obtained
Integral fraction of that with Z=1
ESI MS
Electrostatically-charged nozzle
Solute & solvent
Droplets accelerate away from the tip solvent evaporates
charge concentration become high & Coulombic forces overcome
surface tension resulting in dispersion of the drop into a spray of
smaller droplets.
ESI MS Example 1: peptide
ESI MS Example 2: protein
Genome: the complete spectrum of genetic material of a cell

Proteome: the complete protein composition of a biochemical
system (e.g. all proteins within a cell)

MS determines post-translational modifications
! methylation, phospohrylation, glycosylation, etc
K
a
AH A

+ H
+
- =
+
] log[H pH
[AH]
] ][H [A
K
a
+ !
=
[AH]
] [A
log pK pH
a
!
+ =
a a
logK pK ! =
Henderson-Hasselbalch
1
0
1 1
0
1
, a
RT
G
, a
K ln RT G e K ! = " =
!
2
0
2 2
0
2
, a
RT
G
, a
K ln RT G e K ! = " =
!
a log .
e log
a log
a ln 303 2 = =
( )
a , a , a
o
pK RT . K ln K ln RT G G G ! = " " = " = ! 303 2
1 2
0
1
0
2
a P
pK RT . G ! = ! 303 2
0
Perturbation in the ionization free energy:
Change in pKa in response to local electrostatic environment
Favorable
Coulombic
Interaction:
+
Unfavorable
Coulobic
Interaction:
+ +

Desolvation effect
(transfer from
solvent
to hydrophobic
environment):
+

+ Basic amino acid Acidic amino acid
Arrows denote shifts in pK
a
values with respect to the pK
a
values of
free amino acids in solution
E
electro
=
q
1
q
2
D
medium
r
! =
D
medium
D
vacuum
=
D
medium
"!
0
k = 4#
SI units
D
medium
= 4#!
0
!
Coulombic energy
Coulombs law
Coulombs law describes a pairwise interaction

The potential energy for a single isolated charge in a dielectric
medium is modeled by its self-energy
Dr
e Z Z
V
e
2
2 1
=
0
4!" = D
Coulombs law does not account for shielding of electrostatic
interactions by solvent salt ions ! need more elaborate Poisson-
Boltzmann treatments

Coulombs law is typically used in force field potential energies
S
self
DR
) Ze (
V
2
2
1
=
always positive
R
S
is Stokes radius of ion or molecule
Dielectric constant:
Polarizability of medium
Self-energy
The self-energy of an ion is given by

Consider the work done to bring a small increment of
charge, dq, to the surface of a sphere, already carrying
charge, q
U
self
=
1
4!"
0
"
q
2
2R
S
!U =
! q! ! q
4"#
0
#R
S
U =
1
4"#
0
#R
S
! q d ! q =
1
2
q
2
4"#
0
#R
S
0
q
"
Interactions with environment
Potential energies involving charges &/or dipoles depend on the
polarity of the intervening medium

Charge/dipole interactions are shielded in a polar medium and
thus weakened

Vacuum: least polarizable medium with dielectric constant
#$
0
= 4% 8.85 x 10
-12
C
2
/(J m)
# = 4%
$
0
: vacuum permitivity
$
0
= 8.85 x 10
-12
C
2
/(J m) " SI units
$
0
= 1/(4%) " esu units

Energy: inversely proportional to D

Water: D = 78.5#$
0
@ room T
! =
D
medium
"!
0
SI units:
! =
D
medium
D
vaccum
=
D
medium
4"!
0
!D
medium
= 4"!
0
!
cgs units: 4%$
0
= 1
# = 4% for unit charge
Electrostatic fields
SI units
Charge: C
Energy: J
Distance: m
Potential: V = J C
-1

Capacitance: F = C V
-1

19
12 2 -1
0
23 -1
2
4 -1
0
1.602 10 C
8.854 10 C J m
6.022 10 mol
1.386 10 J m mol
4
c
av
av c
e
N
N e
!
"!
#
#
#
= $
= $
= $
= $
free
pair
AH
f
!"U
neutral
AH
p
"U
free
# $ ##
"U
pair
# $ ##
A
f
%
!"U
charged
A
p
%
+
+
H
+
H
+
"U
&
= 0
For acidic amino acid:
How about for basic amino acid?
How about for desolvation?
Hint for class example