BIO 3601 - Biochemistry

Protein Secondary and Tertiary

Structure, and Folding
Protein Folding and Tertiary Structure

unfolded

folded

From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
 Protein Structure
How do we know that proteins have structure?
 Diffraction

https://www.google.com/search?q=interference&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjNpo6nzbrdAhVpUd8KHZ8fCDQQ_AUIDigB&biw=1748&bih=865
Protein Crystallography
 Protein Structure and Function

Biochemistry: The Molecular

Basis of Life, 5/e
 Protein Representations
Surface All Stick Backbone Stick

RIbbon
 Ribbon Representation of the backbone

http://cmgm.stanford.edu/biochem201/Slides/Protein%20Structure/Alpha%20Helices.JPG
 Chimeric Representations

Ribbon and Stick Ribbon and Surface

Four Examples of Proteins
myoglobin KscA: potassium channel

1BL8
lysozyme beta amyloid

http://wavefunction.fieldofscience.com
 Protein Structure
Primary – The sequence of amino acids in the polypeptide,
held together by covalent bonds (the peptide bond).

Secondary – The local (backbone) arrangement of amino

acids in the three-dimensional structure of the protein, held
together by hydrogen bonds.

Tertiary – The arrangement of secondary structure(s) in the

folded protein, held together by side chain interactions with
water and with one another.

Quaternary – Two or more protein chains forming a

functional complex.
Secondary Structure
 Molecular Geometry

d q
d2 2
H
H H
d1 O O d3
H q1

d1 O d2
q H
H H
O O
H
 Dihedral Angle (G)

B C
A GABCD D
q
d2 2
H
d1 O O d3
H q1
 Dihedral Angle

https://qph.is.quoracdn.net/main-qimg-3d23024fd30f7e49eb5453bd197d2881
 Question
How many atoms do you need the positions of
to define a dihedral angle around a bond?
• A. 1
• B. 2
• C. 3
• D. 4
• E. more than 4
Planar Peptide Bond

Why is the peptide bond planar?

Trans and Cis Peptide Bonds
Proline Peptide Bonds

94% 6%
Dihedral Angles (f,y)

f = C’(i-1) - N(i) – Ca(i) – C’(i)

y = N(i) – Ca(i) – C’(i) - N(i+1)
 Question
• In calculating the phi bond of an amino acid at
position i, you would need the coordinates of
the following atoms:
• A. main chain atoms of the amino acid i
• B. main chain atoms of the amino acid i-1
• C. main chain atoms of the amino acid i+1
• D. both A and B
• E. both A and C
 Question
• In calculating the psi bond of an amino acid at
position i, you would need the coordinates of
the following atoms:
• A. main chain atoms of the amino acid i
• B. main chain atoms of the amino acid i-1
• C. main chain atoms of the amino acid i+1
• D. both A and B
• E. both A and C
Dihedral Angles
 Dihedral Angles

phi, psi: Ramachandran dihedral angles, Omega: peptide bond,

can take a range of values limited to two values: 0 and 180

http://www.cureffi.org/media/2014/09/dihedral-angle-diagram.png
Dihedral Angles
Ramachandran Diagram
Steric Clashes
Ramachandran Diagram
 a-Helix

Hydrogen bond

Side Chains
(f = - 57o, y = -47o)

http://www.chemguide.co.uk/organicprops/aminoacids/basichbond.gif
a - helix
 Helix Dipole

What kinds of amino acids should we expect

to find in the two termini?
 Helical Parameters

Helix
Axis

Repeat
(distance
that Pitch (distance to make 1 turn)
structure
repeats
itself) Rise (1 residue to the next)
a-helix (3.613 helix)

(f = - 57o, y = -47o)

3.6 residues / turn

0.15 nm rise / residue

Pitch = (3.6 residue/turn) x

0.15 nm rise/residue = 0.54 nm

Side Chains
a-Helix Hydrogen Bonding
Helical Hydrogen Bonding

310 helix pi-helix

(-49, -26) (-49, -26)
 310, a, and pi (p) Helices

310 helix a-helix pi-helix

310 helix a-helix pi-helix

Fold helices like a Linus
Ramachandran Diagram
b-strand
Parallel b-sheet
Parallel b-sheet
Antiparallel b - Sheet
 Antiparallel b-sheet

Biochemistry: The Molecular

Basis of Life, 5/e
Mixed b - Sheet
Protein Compaction
b Turns
 Question
Hydrogen bonds stabilize the following
secondary structure:
• A. alpha helix
• B. beta sheets
• C. beta turns
• D. all of the above
 Helix Propensity Scales

DDG: how much a helix is destabilized by the presence of a specific amino acid
compared to alanine
Secondary Structure Propensity

Some aa’s prefer to be in helices,

while others prefer to be in sheets.
Ramachandran Plot

http://www-personal.umich.edu/~lpt/model_files/image003.gif
Ramachandran Plot for Pro

Why does the Ramachandran plot look like this for Pro?
Ramachandran Plot for Gly

Why does the Ramachandran plot look like this for Gly?
 Question
Which of the following statements is true?
• A. All amino acids have the same Ramachandran
plot patterns
• B. All amino acids have the same likelihood to
exist in alpha helices
• C. All amino acids have the same likelihood to
exist in beta sheets
• D. The amino acids have different secondary
structure preferences depending on their
chemical structure and properties
Ramachandran Plot
Soon!
 Tertiary Structure

Amino Acids Beta Strand Alpha Helix Beta Strand

Alpha Helix

Primary Secondary Tertiary Structure

Structure Structure
 Protein Structures

TIM barrel Rossman fold

What are the structural similarities and differences
between these two different proteins?
 Motif
Helix-turn-Helix Coiled-coil Helix Bundle

Motif: regular
combination of
b-a-b b-hairpin b-meander
secondary structural
elements that are
observed in many
proteins
Greek-key b-sandwich
 Example: Helix-Turn-Helix Motif

https://www.google.com/search?q=helix+turn+helix&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjv_Jui8cfdAhUQTN8KHXzeDbkQ_AUIDygC&biw=1748&bih=865#imgrc=uZAKciMq-ylq4M:
 Example: Zinc Finger Motif
X2-Cys-X2,4-Cys-X12-His-X3,4,5-His

Cys

His Zn

Biochemistry: The Molecular

Basis of Life, 5/e
Zinc Fingers Binding to DNA
a/b ‘Folds’
Fold classes

Four general
fold classes
• All alpha
• All beta
• Alpha/beta
• Alpha + beta
 Protein Folding Pathway

Biochemistry: The Molecular Basis of Life, 5/e

Denaturation (Unfolding)
 Anfinsen Experiment
Native Ribonuclease A

Reducing
Agents

Denaturing
Dialysis/Cooling Agents

Unfolded Ribonuclease A
 Question
According to Anfinsen, the structure of a folded
protein is dictated primarily by
• A. the cellular environment
• B. other proteins nearby
• C. the protein’s amino acid sequence
• D. random factors
Micelle
The Protein Hydrophobic Core
 Question
Which of the following is FALSE?
• A. Proteins fold because some amino acid side
chains are hydrophobic while others are polar.
• B. Hydrophobic amino acids are driven to the
interior of a protein to reduce their interaction
with water.
• C. Proteins will fold the same way in a non-
polar solvent like benzene as they do in water.
• D. The DG0 of folding is negative.
 Myoglobin – Surface and Core

Disclaimer: No actual proteins

were harmed in the
preparation of this slide.

http://www.chembio.uoguelph.ca/educmat/phy456/gif/mbcore.gif
 Helical Wheel
8 1 12
15 5
4 16
11 9
18 2
7 13
14 6
3 10 17
Try to fit this sequence in the helical wheel:
Met-Leu-Gln-Ser-Met-Val-Ser-Leu-Leu-Gln-Ser-Leu-Val-Ser-Leu-Ile-Ile-Gln
Helical Polarity
 Question
Which amino acid would you expect to find on
the surface of a globular protein?
• A. Leu
• B. Gly
• C. Glu
• D. Tyr
• E. Trp
 Disulfide Bonds

INSULIN

A simpler case: (Ala-Cys-Thr) : (Gly-Ala-Ala-Cys) connected by a

disulfide bond. Can you draw that peptide?
Protein Folding Puzzle
 Levinthal Paradox
Even for a small protein with only two degrees of freedom per amino
acid, the number of possible conformations is astronomical.

The time required to sample each conformation is also astronomical.

However, experimentally determined folding rates are typically
milliseconds to seconds. Thus, the paradox!

Therefore: random search can not be the mechanism by which

proteins fold into their native conformation.

Consider a protein of 100 amino acids. If each amino acid had only 3
possible structural conformations, the total number of conformations
would be 3100. If we test 1013/sec (which is a lot), we could sample 3 x
1020/yr. Thus, it would take 1027 years to find the right conformation.
 In other words:
Proteins do not fold by random search.

It would take too long.

energy
Protein Folding Landscape

conformation
 Question
When a protein folds into its native conformation,
• A. it goes through all the different
conformations randomly to select the best fold
• B. it goes towards a structure with a high DG0
• C. it forms all favorable intra and intermolecular
interactions that minimizes free energy
• D. all the polar and charged amino acids fold in
the interior of the protein
 An exception: Proteins
without secondary structure

metallothioneins
 Another exception:
Natively unstructured proteins

http://www.nature.com/nrm/journal/v6/n3/images/nrm1589-i1.jpg
 Protein size: smallest possible?

de novo: Tc5b (20 amino acids) de novo: CLN025 (10 residues)

Smallest possible size of a

protein is determined by the
minimal number of amino
acids necessary to form a
stable hydrophobic core

GB1 protein: 58 amino acids Zinc Finger ~ 30 aa/monomer

Image.slidesharecdn.com/technicalslideshow-130315223520-phpapp01/95/technical-slideshow-9-
638.jpg?cb=1363386964 http://www.fccc.edu/research/labs/roder/gb1ribbon.jpg
Large proteins are composed of domains

Fibronectin is a multidomain protein

Lipoxygenase is a single domain protein

 Proteins are (almost) never alone

trimer tetramer
dimer

filament complex capsid

http://www.topsan.org/@api/deki/files/596 https://upload.wikimedia.org/wikipedia/commons/3/3d/1GZX_Haemoglobin.png
 Cell Crowding and Protein Aggregation

Many of our folding experiments (including However, crowded conditions (real cellular
Anfinsen) are done in ideal dilute solutions. conditions) encourage aggregation.

Can the crowded environment have some

effect on protein folding?
http://www.genengnews.com/Media/images/Article/Dilyx_Aggregated_non_aggregated_protein_main7918815399.jpg
 Chaperones

Chaperones prevent aggregation and misfolding

before the protein has a chance to properly fold

Biochemistry: The Molecular

Basis of Life, 5/e
 Alzheimer’s Disease

Aggregation of amyloid fibrils

that form plaques in brain

DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLLVGGVV IA

 Amyloid Folding
• Virtually all proteins can, in
principle, form amyloids
• Amyloid fibrils typically form
from proteins which have
been denatured (unfolded)
and allowed to refold at high
concentrations
• Self association encourages
the further assembly of
amyloid
• This phenomenon poses a
direct challenge to the
Anfinsen hypothesis
 Amyloid Structure

Native Lysozyme Lysozyme Amyloid

 Functional Amyloids

Not all amyloids are bad. Bacteria and yeast produce functional amyloids that coat
their surfaces and facilitate their adhesion to other surfaces. Functional amyloids
have also been found in humans. Still an active area of research.

http://www.amyloidplanet.com/2013/02/functional-amyloid.html
 Prion Diseases

• Mad Cow Disease, Scrapie in goats and sheep,

CJD and Kuru in humans
• severe disruption of brain tissue, which takes
on a disorganized, spongy appearance
• degenerative brain disorder that leads to
dementia (in humans) and eventually death
http://a.abcnews.com/images/WNT/abc_wn_cow_120424_wg.jpg http://learn.genetics.utah.edu/content/molecules/prions/images/BrainSections.jpg
 Prions

Normal Prion Altered Prion Protein

https://microbewiki.kenyon.edu/images/thumb/1/16/R7_prion.jpg/300px-R7_prion.jpg http://www.vce.bioninja.com.au/_Media/prion_med.jpeg
Quaternary Structure
 Question
True or false: All proteins have quaternary
structure.
• A. True
• B. False
 Fibrous Proteins

• provide protection and mechanical support

• have limited amino acid composition
• usually have multiple copies of a defined
sequence repeat
• insoluble and form fibers or filaments
• often (though not always) extensively cross-
linked
• Examples: collagen, keratin, silk fibroin
 Collagens
• the most abundant protein in mammals

• forms a large part of the organic matrix for

bone formation.

• strengthens tendons, ligaments, and skin.

• sheaths muscle tissue.

• adhesion protein in the extracellular matrix.

• makes a fun and tasty dessert!

 Extracellular Matrix

ECM is a complex network of proteins and carbohydrates that fills the space
between cells. ECM holds cells and tissue in place.
 Gelatin
partially hydrolyzed collagen from animals

Disclaimer: City Tech does not endorse a particular flavor of Jell-o.

Collagen primary sequence

Consensus pattern: Gly – Pro – Hyp

Collagen - Left-Handed Helix (3.3)

NOT AN ALPHA HELIX! Why?

Collagen Triple Helix: Tropocollagen
Collagen Structure

Why must there be Gly at every

third position of the helix?
 Question
Collagen has the following secondary structure:
• A. alpha helix
• B. beta sheet
• C. beta turns
• D. all of the above
• E. none of the above
 Osteogenesis Imperfecta (OI)
• Glycine  other amino acid
• Genetic disorder (cannot be
remedied by diet)
• Results in defect in collagen
structure, stability, and
interactions with other proteins
• Brittle bone disease
• Bone fractures, spinal curvature,
and poor muscle tone are
common
• Severe forms are prenatally
lethal
• Loss of hearing
• Blue eye syndrome

https://www.britannica.com/science/osteogenesis-imperfecta
https://www.semanticscholar.org/paper/Perinatal-lethal-osteogenesis-imperfecta-in-a-Thai-Himakhun-
Rojnueangnit/ac1190253e230aa33249eaa4c00ce4e84e1ae0f5/figure/0 /
http://upload.wikimedia.org
Collagen Fibrils

Crosslinking via
side chain
covalent bonding
 Hydroxyproline

Hydroxyproline: allows
the collagen triple helix
to make a sharp turn
 Vitamin C and Scurvy

Scurvy symptoms include malaise, lethargy, bone pain, bleeding,

loose gums, easy bruising

Very common among sailors during the Age of Exploration (16th -

19th centuries)
Why you need your Vitamin C

ferrous ferric

Vitamin C is an enzyme cofactor

a-Keratins
Keratins/Intermediate Filaments

filament structure within the cell for strength & support

http://25.media.tumblr.com/
 a-Keratin Assembly

a- helix

coiled coil

protofilament

filament

Biochemistry: The Molecular

Basis of Life, 5/e
 Coiled – Coil Motif

Sequence: hxxhqxq (h=hydrophobic; q=charged)

 Perms & Hair Straightening

hair fibers (containing keratin) are

held together by disulfide bonds
Silk Fibroin
Silk Fibroin
Tightly packed stacked sheets

Why are Ala and Gly amino acids critical in silk fibroin?
 Question
Which of the following proteins with quaternary
structure contain real alpha helices?
• A. collagen
• B. keratin
• C. silk fibroin
• D. all of the above
• E. none of the above