Fundamentals of Protein

Structure
Traditional Architecture Form Molecular Architecture

fits
function

Wood, brick, nails, glass Materials Amino acids, cofactors

Temperature, earthquakes Environmental Factors Temperature, solubility

How many people? Population Factors # partner proteins, # reactants

How many doors and windows? Portals Passages for substrates and reactants

Spanish, Victorian, Motifs/Styles Conserved domains or protein folds

1950's blocky science building

Julia Morgan Architect Evolution

Levels of Protein Structure
Primary structure = order of amino
acids in the protein chain
Anatomy of an amino acid
Non-polar amino acids
Polar, non-charged amino acids
Negatively-charged amino acids
Positively-charged amino acids
Charged and polar R-groups tend
to map to protein surfaces
Non-polar R-groups tend to be
buried in the cores of proteins
Myoglobin
Blue = non-polar
R-group

Red = Heme
Some R-groups can be ionized
The Henderson-
Hasselblach
equation allows
calculation of the
ratio of a weak acid
and its conjugate
base at any pH

[ HB]
pH = pK '− log
[B− ]
General protein pK’ values
Approximate pK'
Group In a “Typical” Protein
-carboxyl (free) 3 (C-terminal only)
-carboxyl (Asp) 4
-carboxyl (Glu) 4
imidazole (His) 6
sulfhydryl (Cys) 8
1˚-amino (free) 8 (N-terminal only)
-amino (Lys) 10
hydroxyl (Tyr) 10
2˚-amino (Pro)(free) 9 (N-terminal only)
guanido (Arg) 12
Some R-groups can be modified
 Amino Acids Are Joined By
Peptide Bonds In Peptides
- -carboxyl of one amino acid is joined to
-amino of a second amino acid (with
removal of water)
- only -carboxyl and -amino groups are
used, not R-group carboxyl or amino
groups
Chemistry of peptide bond formation
 The Amide bond
Linus Pauling and Corey determined the structure of the peptide
bond by X-ray. O
O-
C
N C +
N
H

40% double bond character. The amide bond or peptide bond

C-N bond is 0.13A shorter than C-N bond. The carbonyl
bond is .02 A longer then those for ketones and aldehydes

Resonance gives 85 kJ•mole-1 stability when bond is planar!!

The peptide bond is planar

This resonance
restricts the number
of conformations in
proteins -- main
chain rotations are
restricted to f and y.
 Primary sequence reveals important
clues about a protein
• Evolution conserves amino acids that are important to protein
structure and function across species. Sequence comparison
of multiple “homologs” of a particular protein reveals highly
conserved regions that are important for function.

• Clusters of conserved residues are called “motifs” -- motifs

carry out a particular function or form a particular structure that
is important for the conserved protein.
motif
DnaG E. coli
small hydrophobic ...EPNRLLVVEGYMDVVAL...
DnaG S. typ
large hydrophobic ...EPQRLLVVEGYMDVVAL...
DnaG
polar B. subt ...KQERAVLFEGFADVYTA...
gp4 T3
positive charge ...GGKKIVVTEGEIDMLTV...
gp4
negative T7
charge ...GGKKIVVTEGEIDALTV...
: : : : * * * : :
Secondary structure = local folding
of residues into regular patterns
The Polypeptide Chain
 Peptide Torsion Angles

Torsion angles determine flexibility of backbone structure

Steric hindrance limits backbone flexibility
Side Chain Conformation
 Sidechain torsion rotamers

• named chi1, chi2, chi3, etc.

e.g. lysine
 chi1 angle is restricted

• Due to steric hindrance between the gamma side chain

atom(s) and the main chain
• The different conformations referred to as gauche(+), trans
and gauche(-)
• gauche(+) most common
 Regular Secondary Structure
Pauling and Corey

Helix Sheet
 Helices
A repeating spiral, right handed (clockwise twist)
helix
pitch = p

Number of repeating units per turn = n

d = p/n = Rise per repeating unit

Fingers of a right - hand.

Several types , 2.27 ribbon, 310 ,  helicies, or

the most common is the  helix.
Examples of helices
 The Nm nomenclature for helices

N = the number of repeating units per turn

M = the number of atoms that complete the cyclic
system that is enclosed by the hydrogen bond.
The 2.27 Ribbon
•Atom (1) -O- hydrogen bonds to the 7th atom in the
chain with an N = 2.2 (2.2 residues per turn)
3.010 helix
•Atom (1) -O- hydrogen bonds to the 10th atom in
the chain with an N= 3.
•Pitch = 6.0 Å occasionally observed but torsion
angles are slightly forbidden. Seen as a single
turn at the end of an  helix.
•Pi helix 4.416 4.4 residues per turn. Not seen!!
 The  helix

The most favorable F and Y angles with little steric hindrance.

Forms repeated hydrogen bonds.
N = 3.6 residues per turn
P = 5.4 Å ( What is the d for an  helix?)
The C=O of the nth residue points towards the N-H of the
(N+4)th residue.

The N H O hydrogen bond is 2.8 Å and

the atoms are 180o in plane. This is almost optimal with
favorable Van der Waals interactions within the helix.
alpha helix
 Properties of the  helix

• 3.6 amino acids per turn

• Pitch of 5.4 Å
• O(i) to N(i+4) hydrogen bonding
• Helix dipole
• Negative f and y angles,
• Typically f = -60 º and y = -50 º
 Distortions of alpha-helices
• The packing of buried helices against other
secondary structure elements in the core of the
protein.
• Proline residues induce distortions of around 20
degrees in the direction of the helix axis. (causes
two H-bonds in the helix to be broken)
• Solvent. Exposed helices are often bent away from
the solvent region. This is because the exposed
C=O groups tend to point towards solvent to
maximize their H-bonding capacity
Top view along helix axis
 310 helix
• Three residues per turn
• O(i) to N(i+3) hydrogen bonding
• Less stable & favorable sidechain packing
• Short & often found at the end of  helices
 Proline helix

Left handed helix

3.0 residues per turn
pitch = 9.4 Å
No hydrogen bonding in the backbone but helix
still forms.
Poly glycine also forms this type of helix
Collagen: high in Gly-Pro residues has this type of
helical structure
Helical bundle
 Helical propensity

How the amino acid tend to participate in helices

The -sheet
• In a -sheet, carbonyl
oxygens and amides form
hydrogen bonds.

• These secondary
structures can be either
antiparallel (as shown) or
parallel and need not be
planar (as shown) but can
be twisted.

• The propensity of a peptide

for forming -sheet also
depends on its sequence.
 Beta sheets
•Hydrogen bonding between adjacent peptide chains.
•Almost fully extended but have a buckle or a pleat.
Much like a „Ruffles” potato chip
Two types
Parallel Antiparallel

N C N C
N C C N
7.0 Å between pleats on the sheet
Widely found pleated sheets exhibit a right-handed twist,
seen in many globular proteins.
Antiparallel beta sheet
Antiparallel beta sheet side view
Parallel beta sheet
Parallel, Antiparallel and Mixed Beta-
Sheets
 beta () sheet

• Extended zig-zag
conformation
• Axial distance 3.5 Å
• 2 residues per repeat
• 7 Å pitch
  turns

• -turns allow the protein backbone to make abrupt turns.

• Again, the propensity of a peptide for forming -turns depends

on its sequence.
 Which residues are common for -
helix, -sheet, and -turn elements?
 Three Dimensional Protein Structures

Confirmation: Spatial arrangement of atoms that

depend on bonds and bond rotations.

Proteins can change conformation, however, most

proteins have a stable “native” conformation.

The native protein is folded through weak

interactions:
a) hydrophobic interaction
b) Hydrogen bonds
c) Ionic bonds
d) Van der Waals attractions
A Denatured protein is unfolded, random dangling, and
often precipitated (cooking egg whites).

The Native conformation is dictated by its amino acid

sequence.

 primary structure is everything.

A one dimensional strand of DNA contains four dimensional data:

height
width
depth
life span!!
 Peptide bonds are planar
Resonance energy depends on bond angle: 180 is max angle  cis
or trans peptide bond.

Most peptide bonds are trans, 10% that follow proline may be cis

Note: differences between bond angles and bond lengths comparing

cis and trans forms.
 Torsion angles
Rotation or dihedral angles

C-N f phi
C-C y psi

When a peptide chain is fully extended the angles are defined as

180 or -180.

At 180 one gets a staggered conformation. (all trans) i.e. ethane

Note: alternating C=O pointing in opposite directions.

When viewed down the N
to C terminus axis, rotation
to the right or clock wise
increases the angle of
rotation.

Must start with the fully

extended form which is
defined as 180o or -180o
Start with fully extended
protein structure Rotate counter
clockwise start at +180o
and decrease angle

Rotate
clockwise start
at -180o and
increase angle

This is C-carbonyl bond or psi angle, Y

 Ethane can exist as staggered or eclipsed conformation

Staggered eclipsed

There is a 12 kJ•mole-1 penalty in energy for an eclipsed

geometry
Bulky amino acid side chains have a much higher energy penalty.
There are a few favored geometries which the protein backbone can fold
If all F + y angles are defined then the
backbone structure of a protein will be
known!! These angles allow a method to
describe the protein’s structure and all
backbone atoms can be placed in a 3d
grid with an x, y, z coordinate.
 Ramachandran plot
If you plot y on the y axis and F on the x axis, you
will plot all possible combinations of F, y.

This plot shows which angles are allowed or which angles are
sterically hindered for poly-l-alanine
 Secondary structure can be defined by f and y
angles
F Y

 helix rt handed -57 -47

 sheet -119 113

  sheet -139 135

310 helix -49 -26

collagen -51 153

Repeating local protein structure
determined by hydrogen bonding
helices and pleated sheets. 12 proteins except for Gly and Pro
Steric hindrance between the amide
nitrogen and the carbonyl

F = -60o and Y = 30o

Ramachandran plot -- shows f and y
angles for secondary structures
Tertiary structure = global folding of
a protein chain
Tertiary structures are quite varied
Quaternary structure = Higher-order
assembly of proteins
Example of tertiary and quaternary
structure - Sir1/Orc1 heterodimer

Example is Sir1/Orc1 complex solved at UW: Hou, Bernstein, Fox, and Keck
(2005) Proc. Natl. Acad. Sci. 102, 8489-94.
 Examples of other quaternary
structures
Tetramer Hexamer Filament

SSB DNA helicase Recombinase

Allows coordinated Allows coordinated DNA binding Allows complete
DNA binding and ATP hydrolysis coverage of an
extended molecule