Protein Structures from an NMR Perspective
Background
–
We are using NMR Information to
“FOLD” the Protein.
–
We need to know how this NMR data
relates to a protein structure.
–
We need to know the specific details of
properly folded protein structures to
verify the accuracy of our own
structures.
–
We need to know how to determine
what NMR experiments are required.
–
We need to know how to use the NMR
data to calculate a protein structure.
–
We need to know how to use the protein
structure to understand biological
function
Protein Structures from an NMR Perspective
Analyzing NMR Data is a Non-Trivial Task!
there is an abundance of data that needs to be interpreted
X
Interpreting NMR Data Requires
Making Informed “Guesses” to
Move Toward the “Correct” Fold
Distance from Correct Structure
Not A Direct Path!
Initial rapid convergence to
approximate correct fold
Correct structure
NMR Data Analysis
Iterative “guesses” allow
“correct” fold to emerge
Protein Structures from an NMR Perspective
What Do We Mean By Informed “Guesses”?
As we will see in detail, analysis of NMR data is commonly ambiguous
But, it represents
a unique structure!
A simple illustration:
Diagonal peak assigned
to Ala 97 CaH
Options:
NOE cross- peak
assigned to Thr 17 CgH
Chemical Shift assignment
of peak consistent with:
Ala 16 CbH
Thr 43 CgH
Ile 36 Cg2H
etc,
1) be conservative and leave the ambiguous peak unassigned
2) Guess the assignment at Ala 16 CbH based on the proximity to assigned Thr 17 CgH
Distance from Correct Structure
Protein Structures from an NMR Perspective
Initial rapid convergence to
approximate correct fold
Iterative “guesses” allow
“correct” fold to emerge
Correct structure
NMR Data Analysis
To progress to the correct protein fold, it is important to make limited “guesses”
Do Not Be Afraid or Hesitant to Make Reasonable “Guesses”!
• if the “guess” is wrong:
 within limits, process is self-correcting  too many guesses are a problem
 the structure combined with the abundance of other correct data will identify the
wrong “guess”
• if the “guess” is correct:
 the assignment will be consistent with the structure  more correct DATA!
 may resolve other ambiguous data
 allow for other “guesses” to further the structure analysis
Protein Structures from an NMR Perspective
What Information Do We Know at the Start of Determining A
Protein Structure By NMR?
Amino Acids (building blocks of protein structures):
Important features of Amino Acids that Impact the Overall Structure of a Protein
Include:






Size
Charge
Polarity
Hydrophobicity
Aromaticity
Conformationally unusual side chains
Protein Structures from an NMR Perspective
What Information Do We Know at the Start of Determining A
Protein Structure By NMR?
Amino Acids (building blocks of protein structures):
Important features of Amino Acids that Impact the Overall Structure of a Protein
Include:

Zwitterion (charge)
The chemistry of amino acids is complicated by
the fact that the -NH2 group is base and the -CO2H
group is an acid. At physiological pH (7.4), an H+ ion
is transferred from one end of the molecule to the
other to form a zwitterion or “salt-like” structure
Protein Structures from an NMR Perspective
Illustration of Zwitterion Characteristics of Amino Acids
from the pH Titration of Alanine
Protein Structures from an NMR Perspective
Polar Amino Acids:
Asparagine, ASN, N
Serine, SER, S
Cysteine, CYS, C
Theronine, THR, T
Glutamine, GLN, Q
Tryptophan, TRP, W
Histidine, HIS, H
(depends on pH)
Tyrosine, TYR, Y
Carbon: gray
Oxygen: red
Hydrogen: white
Nitrogen: light blue
Sulfur: yellow
Protein Structures from an NMR Perspective
Hydrophobic Amino Acids:
Alanine, ALA, A
Phenylalanine, ALA, A
Isoleucine, ILE, I
Proline, Pro, P
Leucine, LEU, L
Valine, VAL, V
Methionine, MET, M
Glycine, GLY, G
Carbon: gray
Oxygen: red
Hydrogen: white
Nitrogen: light blue
Sulfur: yellow
Protein Structures from an NMR Perspective
Charged Amino Acids:
Positive
Arginine, ARG, R
Histidine, HIS, H
(depends on pH)
Lysine, LYS, K
Negative
Aspartate, ASP, D
Glutamate, GLU, E
Carbon: gray
Oxygen: red
Hydrogen: white
Nitrogen: light blue
Sulfur: yellow
Protein Structures from an NMR Perspective
Amino Acid Structures as Part of a Protein Structure:
Knowing the shape and composition
of individual amino acids makes it
easier to identify them as part of a
more complex protein structure
Protein Structures from an NMR Perspective
Amino Acid Structures as Part
of a Protein Structure:
Protein Structures from an NMR Perspective
Venn diagram grouping amino acids according to their properties
Livingstone & Barton, CABIOS, 9, 745-756, 1993
Protein Structures from an NMR Perspective
Name
3-Letter
Code
Single
Code
Relative
Abundance
MW
Alanine
ALA
A
13.0
89
Arginine
ARG
R
5.3
175
Asparagine
ASN
N
9.9
132
Aspartate
ASP
D
9.9
132
Cysteine
CYS
C
1.8
Glutamate
GLU
E
Glutamine
GLN
Glycine
pKa
Residue
Volume (Å3)
Surface Area
(Å2)
Charged, Polar,
Hydrophobic
88.6
115
H
173.4
225
C+
111.1
150
P
4.5
114.1
160
C-
121
9.1-9.5
108.5
135
P
10.8
146
4.6
138.4
190
C-
Q
10.8
146
143.8
180
P
GLY
G
7.8
75
60.1
75
-
Histidine
HIS
H
0.7
155
153.2
195
P, C+
Isoleucine
ILE
I
4.4
131
166.7
175
H
Leucine
LEU
L
7.8
131
166.7
170
H
Lysine
LYS
K
7.0
147
168.6
200
C+
Methionine
MET
M
3.8
149
162.9
185
H
Phenylalanine
PHE
F
3.3
165
189.9
210
H
Proline
PRO
P
4.6
115
112.7
145
H
Serine
SER
S
6.0
105
89.0
115
P
Threonine
THR
T
4.6
119
116.1
140
P
Tryptophan
TRP
W
1.0
204
227.8
255
P
Tyrosine
TYR
Y
2.2
181
193.6
230
P
Valine
VAL
V
6.0
117
140.0
155
H
~12
6.2
10.4
9.7
Protein Structures from an NMR Perspective
Some General Rules Regarding the Distribution of Amino Acids in Proteins:
• Charged residues are hardly ever buried.
► if buried generally involved in “salt-bridge”
• Polar residues are usually found on the surface of the protein, but can be buried.
► if buried generally involved in hydrogen bond
• The inside, or core of a protein contains mostly non-polar residues.
• Non-polar residues are also found on the outside of proteins.
Energetic Cost of Putting Amino
Acid in Interior or Surface of
Protein
Amino Acid Composition of Protein Interior and Surface
Residue
Total
Inside
Surface
-RTln(surface/inside)
Ala
8.7
11.0
7.9
0.20
Arg
3.1
0.4
4.0
-1.34
Asn
5.2
2.0
6.3
-0.69
Asp
6.1
2.2
7.4
-0.72
Cys
2.7
5.4
1.8
0.67
Gln
3.6
1.3
4.5
-0.74
Glu
4.9
1.0
6.2
-1.09
Gly
9.0
9.7
8.6
0.06
His
2.3
2.4
2.2
0.04
Ile
4.9
10.5
3.0
0.74
Leu
6.5
12.8
4.3
0.65
Lys
6.7
0.3
8.9
-2.00
Met
1.5
3.0
0.9
0.71
Phe
3.6
7.7
2.5
0.67
Pro
4.0
2.2
4.7
-0.44
Ser
7.9
5.0
8.9
-0.34
Thr
6.4
4.6
7.1
-0.26
Trp
1.6
2.7
1.3
0.45
Tyr
4.4
3.3
4.8
-0.22
Val
6.6
12.7
4.6
0.61
Totals
5436
1396
4040
Protein Structures from an NMR Perspective
Kyte-Doolittle Hydropathy Ranking of Relative
Amino Acid Hydrophobicity
- Does it make sense for the residue to be on
the protein surface or buried in its core?
- Based on an amalgam of experimental
observations derived from the literature.
- Web page to calculate hydrophobicity plots
for protein sequence
http://fasta.bioch.virginia.edu/o_fasta/grease.
htm
J. Mol. Biol. (1982) 157: 105-132.
Protein Structures from an NMR Perspective
Biological Base Hydrophobicity Scale (Nature (2005):433:377)
- based on the stability of a peptide sequence in a membrane
where n = 0-7
Decreasing stability
- also, variable stability based on position
Protein Structures from an NMR Perspective
Consensus Hydrophobicity Scale (Journal of Chromatography A (2003):1000:637)
Distribution of hydrophobicity rankings
Ala
Arg
Asp
Asn
Cys
Gln
Comparison of four commonly used oil
partioning scales to measure hydrophobicity
ethanol-dioxane, N-methylacetamide, octanolwater, water-cyclohexane
Protein Structures from an NMR Perspective
Consensus Hydrophobicity Scale (Journal of Chromatography A (2003):1000:637)
Distribution of hydrophobicity rankings
Glu
Gly
Met
Phe
His
Ile
Pro
Ser
Leu
Lys
Thr
Trp
Protein Structures from an NMR Perspective
Some General Rules Regarding the Distribution of Amino Acids in Proteins:
• To bury charged or polar residues, residues are probably involved in a “salt bridge” or
hydrogen bond.
Salt Bridge:
Hydrogen Bond:
Salt-bridge
• This minimizes or eliminates the DG transfer energy needed to bury polar or charged
residues
Protein Structures from an NMR Perspective
Propensity of Amino-Acids To Be Present In A Protein’s Active-Site:
• probability of contact with a non-protein atom
positive number means higher than random  likely to be part of active-site
 negative number means lower than random  unlikely to be part of active-site
• does not include protein-protein or protein-peptide interactions
 roles for tryptophan and proline

HIS
CYS
SER
LYS
THR
ASN
ARG
GLN
GLU
ASP
0.360
0.210
0.130
0.100
0.100
0.080
0.055
0.050
0.050
0.045
ALA
MET
ILE
TYR
VAL
GLY
PHE
TRP
LEU
PRO
0.025
0.025
-0.005
-0.040
-0.060
-0.070
-0.120
-0.140
-0.180
-0.200
Holm & Sander, Intelligent Systems for Molecular Biology, 5, 140-146, 1997
Protein Structures from an NMR Perspective
All Amino Acids (except Gly) Have at Least One Chiral Center
• All amino acids in protein are L-configuration
Gly Increases Main Chain Flexibility
• well-conserved during evolution
Branched Side Chains are Stiffer
• Val, Ile, Leu
• chain folding is facilitated (DS is small)
Pro is a Very Rigid Side-Chain
• Also Fixes backbone conformation
• Phi (f) is always -60o
His is Suitable for Enzyme Catalytic Site
• Commonly Found in Protein Active-Site
• pKa (6.0) Near Physiological pH
Cys can form intra or inter-strand bonds
• formation of disulphide bonds between two spatial close Cys
• free Cys can cause problems by forming unwanted cross-linking
Protein Structures from an NMR Perspective
pH Titration of Histidine Side Chain
•
observed pKa is very dependent on the local structure around the histidine
Protein Structures from an NMR Perspective
pH Titration of Histidine Side Chain
•
•
•
Experimentally measure pKa of His by following chemical shift difference of His ring
proton as a function of pH.
Will observe different pKa’s for different His in a single protein based on their local
structure and involvement in protein’s function/activity.
pKa = pH where the observed chemical shift is half-way between protonated and
deprotonated state
pKa
His fully protonated
His fully deprotonated
Protein Structures from an NMR Perspective
pH Titration of Histidine Side Chain
•
•
•
Experimental data for Human Myoglobin
Similar Titrations for Other Side-Chains (Tyr, Glu)
Measure presence of salt-bridge, hydrogen bonds, etc.
Protein Structures from an NMR Perspective
pH Titration of Histidine Side Chain
►
Presence of a protonated side chain affects the local carbon chemical shifts
–
–
Unprotonated:
►
Ca 54.3 ppm
►
Cb 30.7 ppm
Protonated:
►
Ca 53.3 ppm
►
Cb 28.5 ppm
Protein Structures from an NMR Perspective
Spectral properties of amino acids:
• Trp, Tyr, and Phe contain conjugated aromatic rings and absorb UV light.
► Extinction coefficients are:
Trp
5,050 M-1cm-1 (280 nm)
Tyr
1,440 M-1cm-1 (274 nm)
Phe
220 M-1cm-1 (257 nm)
► Extinction coefficients are additive:
Therefore, if a protein contained 3 Tyr and one Trp its extinction coefficient would be:
e =3 x 1,440 + 1 x 5,050 = 9,370
Protein Structures from an NMR Perspective
Basic Amino Acid Nomenclature:
Protein Structures from an NMR Perspective
More Detail Amino Acid Nomenclature:
Each atom is given
a unique identifier.
This includes
equivalent methyl
hydrogens.
Two Versions
of Naming
Convention
31
Protein Structures from an NMR Perspective
Amino Acid 1H NMR Assignments:
Protein Structures from an NMR Perspective
Amino Acid 13C NMR
Assignments:
Protein Structures from an NMR Perspective
NMR Chemical Shifts Exhibit Specific Amino-Acid Trends
–
By combining 2 or more correlated chemical shifts
Protein Structures from an NMR Perspective
Local Protein Structure Affects NMR Chemical Shifts
–
–
Significant Deviations From Random-Coiled Chemical Shifts Are Routinely
Observed
►
Charge state, conformation, covalent modification, etc.
Structure-Based Deviations May be Larger than Residue Based Differences
►
Ring Current Effect
Proximity to Aromatic Rings will have
pronounced affect on NMR Chemical
shifts.
- Affect also depends on spatial
orientation  above/below plane has
different impact than edge on.
- Which amino-acids that are next to
aromatic rings depend on the overall fold
of the protein
Protein Structures from an NMR Perspective
Local Protein Structure Affects NMR Chemical Shifts
►
Hydrogen Bond
–
a dipole-dipole attraction
–
typical ranges:
–
2.4 Å < d < 4.5 Å
–
180o < f < 90o
HN Chemical Shifts and Hydrogen Bond Length
DdN = 19.2dN-3 – 2.3
Wagner et al., JACS, 105, 5948, 1983
Protein Structures from an NMR Perspective
A Number of Amino Acid Hydrogens are Labile and Exchange Readily with Water
–
–
–
Exchange Rate is pH Dependent
As Exchange Increases  NMR Lines Broaden Beyond Detection
Backbone NH is Critical Hydrogen that Exchanges with Water
►
–
Hydrogen Bonds and buried NHs (protected from solvent) decrease Exchange Rate
Reason Why NMR Samples Use low pH Buffers (typically pH 5.0 to 6.5)
NMR Line widths
Increase Exchange Rate
Protein Structures from an NMR Perspective
Overview of Some Basic Structural Principals:
a)
Primary Structure: the amino acid sequence arranged from the amino (N)
terminus to the carboxyl (C) terminus  polypeptide chain
b)
Secondary Structure: regular arrangements of the backbone of the
polypeptide chain without reference to the side chain types or conformation
c)
Tertiary Structure: the three-dimensional folding of the polypeptide chain to
assemble the different secondary structure elements in a particular
arrangement in space.
d)
Quaternary Structure: Complexes of 2 or more polypeptide chains held
together by noncovalent forces but in precise ratios and with a precise threedimensional configuration.
Protein Structures from an NMR Perspective
Primary Structure: linear arrangement of amino-acid sequence
N- Alanine – Glycine – Phenylalanine – … – Tyrosine – Serine – C
Three letter code:
N-Ala-Gly-Phe- … -Tyr-Ser-C
Single Letter code: AGF…YS
Protein Structures from an NMR Perspective
The linear arrangement of amino-acid are joined or connected by the
formation of a peptide bond
The Peptide Bond: chemical
linkage -CO-NH- formed by the
condensation of the amino group
and carboxyl group of a pair of
amino acids to form an amide
bond.
Protein Structures from an NMR Perspective
Important Features of the Peptide Bond:
1)
the bond is always planar.
– Rotation about peptide bond is inhibited
2)
The bond is very stable
– Not generally pH, buffer or temperature labile
•
Boil the sample in very high or low pH to cleave
•
Cleavage more efficient at high pH
– Exception: cleavage occurs at Asp-Pro peptide bond at low pH and
elevated temperatures
•
Half-life at pH 2.5 and 40oC is ~ 50 hrs
Protein Structures from an NMR Perspective
Important Features of the Peptide Bond:
3)
the bond is always trans except for proline
– Cis-proline and trans-proline exhibit unique H-H distances
•
Trans: distance of Ha of residue preceding proline and the proline
Hd is short (<2.5Å)
•
Cis: distance of Ha of residue preceding the proline and the proline
Ha is short (<2.5Å)
Protein Structures from an NMR Perspective
Important Features of the Peptide Bond
4)
Structural Dimensions are well defined
– Bond lengths and bond angles of
peptide bond are known
Protein Structures from an NMR Perspective
General PolyPeptide Nomenclature:
Protein Structures from an NMR Perspective
Amino Acid Structural Nomenclature:
-
Definitions of Torsion Angles
►
Backbone

Phi (f): C’i-1 – Ni – Cai – C’i

Psi (y): Ni – Cai – C’i – Ni+1

Omega (w): Cai-1 – C’i-1 – Ni– Cai
–
constrained to 180o
►
Side-chain

Chi-1 (c1): Ni – Cai – Cbi – Cgi

Chi-2 (c2): Cai – Cbi – Cgi –Cd1i
Note: c1 Ile:Cg1, Ser:Sg, Thr:Og1, Val:Cg1
c2 His: Nd1
Protein Structures from an NMR Perspective
Ramachandran Plot:
-
Peptide Conformation is Defined by f,y diehdrals (w – constrained)
Steric Configurations Limits the Range of f,y diehdrals Available to the
Amino Acid.
►
Pro is more restricted where f is constrained to -60o
►
Gly is less restricted, wider range of f,y diehdrals
Non-Gly/Proline
Allowable
Regions in
f,y space.
Dark Gray
Corresponds
to Most
Favorable
Regions .
Significant
region of f,y
is unallowed
Gly
Protein Structures from an NMR Perspective
Ramachandran Plot:
‒
‒
‒
–
–
If f,y dihedral values were listed for every amino acid
►
Protein Topology is Defined!
Ramachandran considered what combinations of f, y were favorable for each
amino acid
►
Only van der Waals forces were considered.
How many backbone conformations of a 300 residue protein are possible?
►
Only f, y important.
►
f, y need only be given ±15o

i,.e sampled every 30o
►
Consider only minima of Ramachandran plot.
Still Encounter Approximately 10300 conformations!
Levinthal paradoxes:
►
How is the right conformation found?
►
Why are there only ~5,000 protein folds?
Protein Structures from an NMR Perspective
Ramachandran Plot:
‒
Sensitivity of the protein structure to relatively small changes in f, y
Same Number of Amino Acids
f = -57o
y = -70o
f = -57o
y = -47o
f = -74o
y = -4o
Protein Structures from an NMR Perspective
Similar Issues For Side Chain Conformation:
-
Steric considerations define allowable c
Staggered configuration is lowest energy
►
60o, -60o or 180o
Valine c1
180o
60o
-60o
Protein Structures from an NMR Perspective
Protein Structures from an NMR Perspective
Limited Number of Possible Conformers for c1, c2, c3
-
All conformers are not equal energy
Different amino acids have different c energy profile and different population
►
Example Potential Energy Surfaces for Side Chain Dihedrals
•
Still combination of 60o, 180o, or -60o (300o)
Gln/Glu c1 c2 Map
http://spin.niddk.nih.gov/clore/Software/Torsion_angles/protein-tor/protein_side.html
Gln/Glu c2 c3 Map
Protein Structures from an NMR Perspective
►
Example Potential Energy Surfaces for Side Chain Dihedrals
•
Still combination of 60o, 180o, or -60o (300o)
Leu c1 c2 Map
Ile c1 c2 Map
Protein Structures from an NMR Perspective
c2 for Phe, Trp and Tyr are Restricted to 90o or -90o

c1 can still be 60o, 180o or -60o
Phe/Tyr c1 c2 Map
Trp c1 c2 Map
Protein Structures from an NMR Perspective
Primary Structure: Disulphide Bonds
►
►
Distinct regions of the primary polypeptide sequence may be joined by the formation of a
disulphide bond between two spatially adjacent Cysteines.
Disulphide bonds are formed by the oxidation of two cysteine residues to form a covalent
sulphur-sulphur bond which can be intra- or inter- molecular bridges.
- Multiple disulphide bonds are
possible in a protein structure.
- Presence of a disulphide bond(s)
restricts the conformations
available to the protein.
-Disulphide bonds stabilize the
overall protein’s fold by 2.5 - 3.5
kcal/mol.
-Disulphide bond is present in
both folded and unfolded protein.
Probably only contributes
entropically, not enthalpically.
Cysteine
Cysteine
Disulphide bond
Protein Structures from an NMR Perspective
Primary Structure: Disulphide Bonds
►
Restriction of conformational space is more apparent in small protein structures
►
Presence of free Cysteines in the protein structure may cause problems in NMR/X-ray
structural work
–
May cause unwanted interstrand cross-linking  aggregation/solubility issues
–
Use reducing agents (DTT, 2-Mercaptoethanol) or mutate Cys to Ser.
Protein Structures from an NMR Perspective
Primary Structure: Disulphide Bonds
►
Geometry of a disulphide bond
–
–
–
Sg – Sg covalent bond length of 2.08Å
Defined by 5 dihedral angles
Two main types:

Left-handed: c1 -60o c2 -60o c3 -85o c2’ -60o c1’ -60o Ca-Ca distance
5.88±0.49Å

Right-handed: c1 -60o c2 +120o c3 +99o c2’ -50o c1’ -60 Ca-Ca distance
5.07±0.73Å
Protein Structures from an NMR Perspective
Primary Structure: Disulphide Bonds
►
Presence of a disulphide bond affects the local carbon chemical shifts
–
–
Reduced:
–
Ca 56.9 ppm
–
Cb 28.9 ppm
Oxidized:
–
Ca 54.05 ppm
–
Cb 42.25 ppm
Cysteine
Cysteine
Disulphide bond
Protein Structures from an NMR Perspective
What Information Do We Know at the Start of Determining A
Protein Structure By NMR?
Effectively Everything We have Discussed to this Point!
The primary amino acid sequence of the protein of interest.
► All the known properties and geometry associated with each
amino acid and peptide bond within the protein.
► General NMR data and trends for the unstructured (random
coiled) amino acids in the protein.
 The number and location of disulphide bonds.
► Not Necessary  can be deduced from structure.
Protein Structures from an NMR Perspective
Secondary Structure: regular arrangements of the backbone of the polypeptide chain
without reference to the side chain types or conformation
Major Types of Secondary Structure Elements:
► helices
 a-helix
 310helix
 p-helix
► b-strands
 parallel
 anti-parallel
► Turns
 b turns
 types I,I’,II,II’,III,III’,Via,VIb
 g turns
Inverse
► Other or random coil
Assigning the Secondary Structure is the First Stage of Determining an NMR Protein Structure
Protein Structures from an NMR Perspective
Secondary Structure: Helices
-
Helix Nomenclature
Protein Structures from an NMR Perspective
Secondary Structure: Helices
Secondary structures are typically distinguished by f,y values and hydrogen bonding
pattern
-
Radius (Å)
(backbone)
Atoms
in Hbonded
loop
(A)
H-bond
pattern
(CO, HN)
i,i+4
Structure
f
y
Residues
per
helical
turn
a-helix
-57
-47
3.6
1.5
5.4
2.3
13
310-helix
-74
-4
3.0
2.0
6.0
1.9
10
p-helix
-57
-70
4.4
1.1
5.0
2.8
16
Helical
Rise
r(Å)
Helical
Pitch
p(Å)
i,i+3
i,i+5
Protein Structures from an NMR Perspective
Secondary Structure: Helices
-
Secondary structures are typically distinguished by f,y values and hydrogen bonding
pattern
Protein Structures from an NMR Perspective
Secondary Structure: Helices
‒
‒
‒
‒
‒
‒
a-helix – most common helix found in protein structures  most thermodynamically stable
►
31% of secondary structure elements
Right-handed twist to helix.
Helix Dipole
~ 85% of helices are distorted (f,y ≠ -60o)
Amino-acid preference in a-helix
Side-chains on the Surface of Helix
Protein Structures from an NMR Perspective
Secondary Structure: Helices
‒
Amino Acid Preference for a-Helix
a-Helix Propensity
(larger number better)
Ala: 1.489
Arg: 1.224
Asn: 0.772
Asp: 0.924
Cys: 0.966
Gln: 1.164
Glu: 1.504
Gly: 0.510
His: 1.003
Ile: 1.003
Leu: 1.236
Lys: 1.172
Met: 1.363
Phe: 1.195
Pro: 0.492
Ser: 0.739
Thr: 0.785
Trp: 1.090
Tyr: 0.787
Val: 0.990
Protein Engineering 1:289-294(1987).
J. Mol. Biol. (2004) 337, 1195–1205
Protein Structures from an NMR Perspective
Secondary Structure: Helices
‒
‒
Amphipathic a-helix
have a polar and a non-polar side
►
‒
hydrophobic residues are regularly spaced three or four position apart in a
linear sequence.
plays a crucial role in
►
►
►
►
►
helix-helix interaction
interaction of small peptides that have a helical conformation
interaction with membranes
air-water interfaces
self-assembly processes
Helical wheel representation of amphipathic a-helix
leucine zipper
Protein Structures from an NMR Perspective
Secondary Structure: Helices
‒
‒
Amphipathic a-helix
have a polar and a non-polar side
Amphipathic a-helix intereacts with membrane
Protein Structures from an NMR Perspective
Secondary Structure: Helix Dipole
►
►
►
CO - HN H-bonds are almost parallel with the helix axis  H-bond dipoles reinforce in the
helix to form helix dipole
Helix dipole (+ end towards N-terminal)

capping by hydrogen bonding to NH and CO groups at the N- and C-termini

charge-dipole interactions
charged side chains form stabilizing interactions with the helix dipole.
Protein Structures from an NMR Perspective
Secondary Structure: Helix Dipole
►
Residues preferred at N- and C-terminus of an a-helix
Protein Science (1995), 4:1325-1336.
Protein Structures from an NMR Perspective
Secondary Structure: Helices
‒
~ 85% of helices are distorted (f,y ≠ -60o)
►
radius of curvature > 90Å
►
deviation of axis from straight line is ≥ 0.25Å.
‒
Distortions caused by:
►
A substantial amount of all 310-helices occur at the ends of a-helices.
►
p-helixes also occur at the ends of a-helices.
►
Packing of buried helices against other secondary structural elements in the core
of a protein can lead to distortions since the side chains are on the surface of
helices.
►
Proline residues induce distortions of around 20o in the direction of a helix.

Proline causes 2 hydrogen bonds in the helix to be broken.

Helices containing proline are usually long because shorter helices would
be destabilized.
►
Exposed helices are often bent away from the solvent.

CO form H-bonds with solvent
Protein Structures from an NMR Perspective
Secondary Structure: Helix Length
‒
‒
‒
Average Length of a-helix is 10 residues
One helical turn requires ~4 residues  defines minimal length
Helix Nomenclature: ...-N''-N'-Ncap-N1-N2-N3-............-C3-C2-C1-Ccap-C'-C''-...
►
Ncap : N-terminus of helix, Ccap: C-terminus of Helix
Stability of Helix Length
Depends on Relative Spatial Orientation of Ncap, Ccap, etc
Position of C-cap relative to N-cap
in function of length.
The good length are black circle,
the bad length are white circle.
The N-cap is a cross.
Position of C2 relative to N-cap in
function of length.
The good length are black circle,
the bad length are white circle.
The N-cap is a cross.
Protein Structures from an NMR Perspective
Secondary Structure: 310-helix and p-helix
–
–
310-helix is rare
►
Only 3.4% of helical residues.
►
Found at end of a-helix.
►
Dipoles not aligned as in a-helix.
►
3 residues per turn & 10 atoms enclosed in
ring formed by each hydrogen bond.
►
CO forms H-bond with NH 3 residues along
chain (i,i+3)
p-helix is extremely rare
►
Found at end of a-helix
►
f,y at edge of allowed region of
Ramachandran plot
►
t (N-Ca-C') angle is 114.9o larger than
standard 109.5o
►
Larger radius causes axial hole too small for
solvent
►
Side-chains less staggered than a-helix
Protein Structures from an NMR Perspective
Secondary Structure: b-strands
–
b-sheet is an abundant secondary structure  25% of globular proteins
►
►
►
►
►
b-strands adopt an extended structure with an average length of 6 residues
Single b-strands are not stable.
If the b -strand contains alternating polar and non-polar residues  amphipathic b -sheet.
b-strands occur in association with other strands to form b-sheets.

Strands can be parallel: NC or anti-parallel: NC
NC
CN

b-strand has right-handed twist (0-30o per residue)
Hydrogen bonding occurs between strands

H-bond geometry is different between parallel and anti-parallel strands
Structure
f
y
Residues per repeat
Rise r(Å)
Pitch p(Å)
Parallel b-strand
-119
113
2.0
3.2
6.4
Anti-parallel b-strand
-139
135
2.0
3.4
6.8
rise
Rise – distance between
adjacent residues
Pitch- distance between
repeat structure
pitch
Protein Structures from an NMR Perspective
Secondary Structure: b-Sheets
-
Secondary structures are typically distinguished by f,y values and hydrogen bonding
pattern
b
a
Protein Structures from an NMR Perspective
Secondary Structure: b-strands
–
anti-parallel b-sheet
►
►
Left-handed twist (~25o)
Majority of bulges occur in anti-parallel b-sheets
Note: alternating
spaced H-bonds
b-strand II
Hydrogen bonds
between NH (blue)
and CO (red)
C-terminus
N-terminus
b-strand I
H-bond length
2.9±0.3Å
Protein Structures from an NMR Perspective
Secondary Structure: b-strands
–
b-bulge
►
hydrogen-bonding of two residues from one strand with one residue from another
strand
Bulge
Hydrogen bonds from residue 33 to both residues 41 and 42
Protein Structures from an NMR Perspective
Secondary Structure: b-strands
–
parallel b-sheet
►
►
►
Less twisted than anti-parallel b-sheets
Less likely to have a bulge compared to anti-parallel b-sheets (only ~ 5%)
Hydrogen bonds are not perpendicular to individual strands

Has macrodipole that is ~5 times less than average a-helix dipole
b-strand II
Hydrogen bonds
between NH (blue)
and CO (red)
N-terminus
C-terminus
b-strand III
b-strand I
Note: Individual strands that comprise a sheet do not need to be sequentially related or the same size
Protein Structures from an NMR Perspective
Secondary Structure: b-Sheets
–
–
–
–
–
b-sheet can continue in both directions.
►
Most b-sheets have < 6 b-strands with an average of 6 residues per strand.
►
H-bonds are 0.1Å shorter than a-helix
b-sheets can be all parallel, all anti-parallel or mixed.
Formed from strands that are very often from distant portions of the polypeptide sequence.
Lengths of individual strands can vary.
►
Do not need to be of uniformed length
Most b-sheets exhibit a left-handed twisted (~25o).
►
results from a relative rotation of each residue in the strands by 30 o per amino acid in a
right-handed sense.
Protein Structures from an NMR Perspective
Secondary Structure: b-sheet
‒
Amino Acid Preference for b-Sheet
►
Hydrophobic and steric effects are unimportant
►
inductive effect largely determines the beta-sheet propensities

amino acid side chains shielding of the Ca nucleus
►
No capping preference has been identified to date
b-Sheet Propensity
(larger number better)
Ala: 0.79
Arg: 0.94
Asn: 0.66
Asp: 0.66
Cys: 1.07
Gln: 1.00
Glu: 0.51
Gly: 0.87
His: 0.83
Ile: 1.57
Leu: 1.17
Lys: 0.73
Met: 1.01
Phe: 1.23
Pro: 0.62
Ser: 0.94
Thr: 1.33
Trp: 1.24
Tyr: 1.31
Val: 1.64
Protein Structures from an NMR Perspective
Secondary Structure: Turns
‒
Short and tight structural regions that connect other secondary structure
elements
►
Comprised of 3 to 5 residues
►
Allows the peptide chain to reverse directions

Therefore, Proline and glycine are prevalent in turns

Connect adjacent b-strands
►
Reverse turns occur mainly on the surface

Therefore, charged residues are prevalent in turns
►
Two common turns:

b-turns

More common turn

Four consecutive residues, two do not form H-bonds

Carbonyl of one residue is H-bonded to amide proton of a
residue three residues away

Distance < 7 Å between the Ca atoms of residue i and i+3

Nine types of b-turns differ by f, y of i+1 and i+2 residues

Types I’, II’, III’ are mirror images of Types I, II, III

Type III b-turns may be considered as short regions of 310helix

g-turns

Very tight turn

Three consecutive residues, one does not H-bond.
Protein Structures from an NMR Perspective
Secondary Structure: Turns
-
-
Secondary structures are
typically distinguished by f,y
values and hydrogen bonding
pattern
Some preferred residues are
indicated, bold are most
significant
i+ 1
i+ 2
f
y
f
y
R(i)
R(i+1)
R(i+2)
R(i+3)
Type I
-60
-30
-90
0
C, P, S,
H, N, D
P, S, E
T, S, N,
D
G
Type I’
60
30
90
0
Y
G, H, N,
D
G
K
Type II
-60
120
80
0
Y, P
P, K
G
C, S, K
Type II’
60
-120
-80
0
G
Type III
-60
-30
-60
-30
Type III’
60
30
60
30
Turn
b-turns
Type IV
G
G
Deviations of more than 40o of the above
Type V
-80
80
80
-80
Type V’
80
-80
-80
80
Type VIa1
-60
120
-90
0
cis-P
Type VIa2
-120
120
-60
0
cis-P
Type VIb
-135
135
-75
160
cis-P
Type VIII
-60
-30
-120
120
Turn
70 to 85
-60 to -70
Inverse Turn
-70 to -85
60 to 70
g-turns
Protein Structures from an NMR Perspective
Secondary Structure: b-turns
‒
Illustration of the Type I & II b-turns and mirror images
Hydrogen Bond
Protein Structures from an NMR Perspective
Secondary Structure: g-turns
‒
Illustration of the classical and inverse g-turn
Hydrogen Bond
Protein Structures from an NMR Perspective
Secondary Structure: Turns
‒
Amino acids preference for turns
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
arrangements of two, three or more consecutive secondary structures
►
►
►
a-helices or b-strands
Common features in many different proteins
Completely different amino acid sequences
(a) βαβ - two parallel strands of β-sheet
connected by a stretch of α-Helix
(b) αα - two anti-parallel α-helices
(c) β meander - an anti-parallel sheet formed
by a series of tight reverse turns connecting
stretches of a polypeptide chain
(d) Greek Key –a repetitive super-secondary
structure formed when an anti-parallel sheet
doubles back on itself
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
►
2 or more a-helices
Contains a heptad repeat (H –hydrophobic; P – polar):
a b c d e f g
(H P P H P P P)n
–
–
–
Leucine zippers – leucine in d position
N is ≥ 3
Knob (a and d) into hole interactions
Knobs
Moutevelis and Woolfson (2009) J. Mol. Biol. 385:726
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
Periodic Table
–
–
–
–
►
Number of coils increase to right
–
–
►
Leucine zippers – leucine in d position
N is ≥ 3
Knob into hole interactions (KIH)
Population and percentage of occupancy
Circle helix, lines KIH, grey hydrophobic
core
Population and percentage of occupancy
below each architecture.
Complexity increases down column
–
–
Helix shared between two helix coiled
coils
Interface between 2 or more coiled coils
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
–
Coiled coils
Diversity of structures
Kohn et al. (1997) J. Biol. Chem. 272:2583
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
Packing angle (W) and axial separation
–
–
Angle between two helices
Shortest distance between the two helices
Walther et al. (1998) PROTEINS 33:457
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
Average axial separation differ for transmembrane and soluble coiled coils
–
–
–
Solution: 9.6 Å
Transmembrane: 9.0 Å. Two clusters ate 7.3 Å and 10.8 Å
Transmembrane coiled coils are more compact contain shorter amino acids (Gly)
Eilers et al. (2000) PNAS 97:5796
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
Average axial separation varies linearely with amino-acid volumes
–
Size (volume) of residues at helix contact
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
Packing angle (W)
–
distribution
Preferential angles are:
W ~ -45o
W ~ +23o
W ~ +75o
Bowie (1997) Nature Structural Biology 4:915
Protein Structures from an NMR Perspective
SuperSecondary Structure:
–
Coiled coils
►
Packing angle (W)
–
–
Depends on geometry of hydrophobic residues
Steric compatibility alone defines packing angle
Heptad repeat
Side by side
11-residue repeat
long
Heptad repeat
face to face
11-residue repeat
normal
W ~ 20o
W ~ 20o
W ~ 0o to -10o
W ~ -30o to -40o
Efimov (1999) FEBS Letters 463:3
Protein Structures from an NMR Perspective
Tertiary Structure:
–
the three-dimensional folding of the polypeptide chain to assemble the different
secondary structure elements in a particularly arrangement in space
Protein Structures from an NMR Perspective
Tertiary Structure:
–
–
Periodic table of Protein Folds
Set of idealized structures

Experimental structures are compared to idealized set to find best match and
classification
Basis Set- most biologically important protein
structures are derived from these idealized structures
a-helix
b-strand
Taylor (2002) Nature 416:657
Protein Structures from an NMR Perspective
Tertiary Structure:
–
–
Periodic table of Protein Folds
Set of idealized structures – looking edge on

4- layers thick
Small Circles - helix
Bars - b-sheet
Arc – curved b-sheet
Open circle – b-barrel
Protein Structures from an NMR Perspective
Tertiary Structure:
–
the three-dimensional folding of the polypeptide chain to assemble the
different secondary structure elements in a particularly arrangement in space
►
►
~800 unique folds have been identified
1,000 – 5,000 protein folds are predicted
SCOP: Structural Classification of Proteins. 1.75 release http://scop.mrc-lmb.cam.ac.uk/scop/index.html
38221 PDB Entries (23 Feb 2009). 110800 Domains. 1 Literature Reference
(excluding nucleic acids and theoretical models)
Class
Number of folds Number of superfamilies Number of families
All a proteins
All b proteins
a and b proteins (a/b)
a and b proteins (a+b)
Multi-domain proteins
Membrane and cell
surface proteins
Small proteins
Total
284
174
147
376
66
507
354
244
552
66
871
742
803
1055
89
58
110
123
90
1195
129
1962
219
3902
Protein Structures from an NMR Perspective
Tertiary Structure:
Family: Clear evolutionarily relationship
Proteins clustered together into families are clearly evolutionarily related. Generally, this means that pairwise
residue identities between the proteins are 30% and greater. However, in some cases similar functions and
structures provide definitive evidence of common descent in the absense of high sequence identity; for
example, many globins form a family though some members have sequence identities of only 15%.
Superfamily: Probable common evolutionary origin
Proteins that have low sequence identities, but whose structural and functional features suggest that a common
evolutionary origin is probable are placed together in superfamilies. For example, actin, the ATPase domain of
the heat shock protein, and hexakinase together form a superfamily.
Fold: Major structural similarity
Proteins are defined as having a common fold if they have the same major secondary structures in the same
arrangement and with the same topological connections. Different proteins with the same fold often have
peripheral elements of secondary structure and turn regions that differ in size and conformation. In some cases,
these differing peripheral regions may comprise half the structure. Proteins placed together in the same fold
category may not have a common evolutionary origin: the structural similarities could arise just from the
physics and chemistry of proteins favoring certain packing arrangements and chain topologies.
Protein Structures from an NMR Perspective
Tertiary Structure:
–
Classifying protein structures is not straightforward or definitive
►
CATH v3.2
Multiple equally valid approaches
http://www .cathdb.info/
Mainly a (1)
5
386
875
2917
37038
Mainly b (2)
20
229
520
2618
43881
Mixed a b (3)
14
594
1113
6183
90029
Few
Secondary
Structures (4)
1
104
118
208
2588
40
1313
2626
11926
173536
Totals
CATH assigns each protein domain to a four number code based on its class
(C), architecture (A), topology (T), and homologous super family (H).
Example:
chain A from PDB ID: 1kbl is assigned a CATH code of 1.20.80.30
class: ................................................... 1 – mainly alpha
architecture: ........................................ 20 – Up-down bundle
topology:............................................. 80 – Acyl-CoA Binding Protein
homologous super family: ................. 30 – no description
Protein Structures from an NMR Perspective
CATH is a novel hierarchical classification of protein domain structures, which clusters proteins at four
major levels:
Class ( ) derived from secondary structure content, is assigned for more than 90% of protein structures automatically.
Architecture ( ) describes the gross orientation of secondary structures, independent of connectivities, is currently assigned
manually.
Topology ( ) clusters structures according to their toplogical connections and numbers of secondary structures are made by
sequence and structure comparisons.
Homologous super family ( ) cluster proteins with highly similar structures and functions are made by sequence and
structure comparisons.
Other Levels:
Sequence Family ( ): cluster proteins based on sequence identity ≥35%, nearly always have identical structure
Non-Identical ( ) : cluster proteins based on sequence identity ≥95%
Identical ( ): numerous cases where the protein structure based on the identical sequence has been deposited into the PDB.
Domain ( ): semi-independent folding unit
Protein Structures from an NMR Perspective
Tertiary Structure: Some Common Examples
Mainly a (4-helix bundle)
Mixed a/b (a/b-barrel)
Mainly b (b-sandwich)
Minimal Secondary Structure (Kringle Domain)
Protein Structures from an NMR Perspective
Tertiary Structure: Continuity of Fold Space
Common Structure Core
(dark grey) for 3.40.50.300
But, different members also have
different structural elements (clear)
Making distinct fold classification is difficult and
problematic because of fold overlap and divergence
Cuff et al. (2009) Structure 17:1051
Protein Structures from an NMR Perspective
Tertiary Structure:
–
–
Similarity to existing protein fold increases likelihood of accuracy of structure
determination
►
All structural folds are not known
►
May have a novel fold
Structures generally not observed
►
Knot structure are highly unlikely
►
Previously thought not to exist.

View a knot in a structure to be suspect

Need to be indisputably verified by experimental data
Knot Structure in acetohydroxy acid isomeroreductase
simple trefoil knots
Protein Structures from an NMR Perspective
Tertiary Structure:
–
RCSB Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do)
►
Database of all known NMR and X-ray Protein structures (> 93,970 structures)
►
Includes DNA, RNA, small molecule ligands and peptide complexes
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
–
Levinthal paradox (Journal de Chimie Physique et de Physico-Chimie Biologique (1968) 65: 44–45)
►
Chain with 101 amino acids
►
three permissible conformations per residue pair
►
3100 or 5x1047 configurations!
►
Sample new configuration at the rate of 1013/sec it will take 1027 years
►
Proteins do not fold by random search
Anfinsen dogma (Science (1973) 181: 223)
►
Thermodynamic hypothesis – native structure is the one in which DG of the whole
system is the lowest

►
Native conformation determined by the totality of interatomic interactions
Amino acid sequence determines native structure
Regions of native
structure form that act
as nucleation sites
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Apply Energy Bias to Search for Native Structure (PNAS (1992) 89: 20-22)
►
N – number of amino acids, ko & k1 rate to form incorrect and correct local
conformation
►
Rates ~ 109 s-1


Simple model, depends on choice of magnitude and ratio of rates
Changes in k1 shifts the graph vertically
DG of typical globular
protein is ~ -5 to -15
kcal/mol
1 kT = 0.593 kcal/mol
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Water is a Poor Solvent for Unfolded or Denatured Proteins
►
If wasn’t true proteins wouldn’t fold!
►
Specifically, water is a poor solvent for protein backbone
–
Protein Energetics
►
Hydrophobic effect contributes ~ -8 kJ/mol per buried residue or ~ -1.912 kcal/mol

►
Hydrogen bonding contributes ~ controversy



►
Destabilizing: DG = +3.1 kcal/mol (Biochemistry (1990) 29: 7133)
Partially stabilizing and destabilizing DG = -1 kcal/mol (J. Mol. Biol. (1999) 293:283)
Important driving force 40 cal/mol per residue (J. Biol. Chem. (2003) 278:31790)
Intramolecular hydrogen bonds are marginally
favored over water:backbone hydrogen bonds


►
-0.9 kcal/mol per buried C atom
Generally accepted view: DG = -1 kcal/mol
Different dielectric values
Difficult to measure hydrogen bond energies
heavily dependent on model.
Chem. Rev. (1997), 97:1251-1267.
System
Dielectric
Alkanes
1-2
Alkenes
2-3
Dry protein
2
Slightly wet protein
4
water
80
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
TMAO
Hydrogen bond contribution to native structure
►
Osmolytes interact with protein backbone
betaine
sucrose
trehalose
sarcosine
sorbitol
proline glycerol
urea guanidine
Colored by water polarity
Larger energetic effect for
transfer backbone into
stabilizing osmolyte
PNAS (2006), 103:13997-14002.
Chem. Rev. (1997), 97:1251-1267.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Buried Salt-Bridges in Folded Protein Structures
►
On average, buried salt bridges are energetically favorable





►
►
►
Note: high desolvation energy and high standard deviation
dslv – unfavorable desolvation energies
brd – favorable bridge energy due to electrostatic interactions of side-chains
prt – favorable electrostatic interaction of salt-bridge with rest of protein
assoc – does not consider electrostatic interaction with rest of protein
66 buried salt bridges and 156 exposed salt bridges
190 out of 222 are stabilizing (55 buried salt bridges)
32 out of 222 are destabilizing (11 buried salt bridges)
J. Mol. Biol. (1999), 293:1241-1255.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Salt Bridges Tend to be Tightly Clustered
►
Most are separated by 5 or less residues
J. Mol. Biol. (1999), 293:1241-1255.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Ionizable residues Tend to be on the Outer Protein Surface
►
Can interact with water
►
Interior of the protein is hydrophobic
►
Are rarely buried – high DG of solvation

2.8 times as many buried acids as bases
1% DDGrxn
> 5 DpK
Defined as buried
20% ASA
Accessible surface area (ASA)
compared against DDGrxn
6.8 kcal/mol ~ DpKa of 5 pH units
JMB (2005), 348:1283-1298.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Ionizable residues Tend to be on the Outer Protein Surface
►
Can interact with water
►
Interior of the protein is hydrophobic
►
Are rarely buried – high DG of solvation

2.8 times as many buried acids as bases
> 5 DpK
Distribution of buried charged
residues decreases rapidly.
6.8 kcal/mol ~ DpKa of 5 pH units
JMB (2005), 348:1283-1298.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
When an apolar residue is replaced by a charged residues
►
Increased tendency to be exposed and solvated
►
Involved in a salt-bridge or hydrogen bond interaction
►
At pH 7, still ionized
JMB (2005), 348:1283-1298.
PROTEINS (2003), 53:783-791.
Plot of average depth of buried apolar residues, averaged for every 1Å interval
of depth, and the normalized occurrence for the cases when they are
substituted by buried charged residues and by exposed charged residues.
Protein Structures from an NMR Perspective
Protein Stability and Folding: (PNAS (2008), 105:17784-17788.)
–
Recent Analysis Suggest Proteins have a High Tolerance for Buried Ionizable Residues
►
25 internal positions in S. aureus nuclease were substituted with Arg, Lys Glu & ASP


►
No loss in activity or changes in structure
86 of 87 changes were destabilizing
pKa of buried ionizable residues were shifted to be neutral at physiological pH
Decrease in stability of mutant
versus wild-type protein
DG S. aureus nuclease – 12.5 kcal/mol
Difference in the above
stability curves
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Recent Analysis Suggest Proteins have a High Tolerance for Buried Ionizable Residues
►
25 internal positions in S. aureus nuclease were substituted with Arg, Lys Glu & ASP


►
No loss in activity or changes in structure
86 of 87 changes were destabilizing
pKa of buried ionizable residues were shifted to be neutral at physiological pH86 of

Protein interior is less polarizable and polar compared to water.
Difference in the thermodynamic
stability curves
Similarity between the
two curves implies a
pKa shift of Lys
Thin lines are Lys titration curves
for pKa of 6.0 and 10.4.
Solid line is the area between the
two titration curves
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Transfer of non-polar compound into
polar solvent is highly unfavorable.
–
Protein Thermodynamics Determined by
Chain Length
►
Surface area increases with number
of residues
►
Burying hydrophobic residues is
favorable
Chem Rev. (1997), 97:1251-1267.
Biochemistry (1990), 29:7133-7155.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Protein Thermodynamics Determined by Chain Length
►
All thermodynamic parameters increase linearly with number of residues
►
Protein stability increase with number of residuesc
Heat Capacity (Cp)
Enthalpy (H)
Entropy (S)
Chem Rev. (1997), 97:1251-1267.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Formation of Secondary Structure Elements are Favored as Compactness Increases
►
Radius of Gyration – measure of structure compactness
where N is the number of atoms and r is the
position vector
Or
where N is the number of residues
–
Unique Native Conformation Corresponds to Optimal Arrangement of Hydrogen Bonds
Biochemistry (1990), 29:7133-7155.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Mechanism of Protein folding
►
Hydrophobic collapse
►
Re-organization into native state to optimize alignment of hydrogen bonds
unfolded
Structural
collapse
Blue spheres indicate
fully solvated regions
(water molecules)
Native
Curr. Opin. Struct. Biol. (2004), 14:70-75.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Mechanism of Protein folding
►
Hydrophobic collapse
►
Re-organization into native state to optimize alignment of hydrogen bonds
Curr. Opin. Struct. Biol. (2004), 14:70-75.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Mechanism of Protein Folding, Protein Assocation and Aggregation are Closely Related
►
Aggregation propensity best prediction of protein-protein interfaces
►
Chaperone’s play an important role in folding a sub-class of proteins
Nat. Struct. Mol. Biol. (2009), 16:574-581.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Aggregation propensity best prediction of protein-protein interfaces
►
Amino acids physico-chemical properties influence aggregation
Hydrophibicity
Charge
a-helix & b-strand propensity
Nature (2003), 424:805-808.
Protein Structures from an NMR Perspective
Protein Stability and Folding:
–
Aggregation propensity best prediction of protein-protein interfaces
►
Sequence based prediction of aggregation
Increasing aggregation
propensity
JMB (2005), 350:379-392.
Protein Structures from an NMR Perspective
Quaternary Structure:
–
Complexes of 2 or more polypeptide chains held together by noncovalent forces but in
precise ratios and with precise three-dimensional configuration
►
Homo- multiple repeats of the same protein
►
Hetero- combinations of different proteins
Homo-multimers
Hetero-multimers