Important biological functions of proteins
1. Enzymes, the biochemical catalysts
2. Storage and transport of biochemical molecules
3. Physical cell support and shape (tubulin, actin, collagen)
4. Mechanical movement (flagella, mitosis, muscles)
5. Decoding cell information (translation, regulation of gene expression)
6. Hormones or hormone receptors (regulation of cellular processes)
7. Other specialized functions (antibodies, toxins etc)

Single monomers polymer
2 to ~50 aa all “ l ” building blocks neurotransmitters hormones
polymer
50+ aa well defined structures enzymes receptors

Proteins are composed of 20 common amino acids
Each amino acid contains:
(1) Carboxylate group
(2) Amino group
(3) Side chain unique to each amino acid
Drawn at very acidic pH (pH=1)

At what pH will a carboxylic acid be deprotonated? pH > pK a pH > 2.5
At what pH will an amine be protonated?
pH < pK a pH < 9
What will the ionization state of an amino acid be at neutral pH?
It depends on the specific amino acid and its sidechain pK a pK a
9-10 pK a
2-3
Amine Carboxylate
5

Amino Acid pka & pI
Amino Acid pKa
C
Glycine
Alanine
Valine
Leucine
Isoleucine
Proline
Serine
Threonine
Cysteine
Methionine
Asparagine
Glutamine
Phenylalanine
Tyrosine
Tryptophan
Lysine
Arginine
Histidine
Aspartate
Glutamate
2.2
2.2
2.2
2.5
2.2
1.8
1.8
2.0
2.1
2.4
2.4
2.3
2.3
2.3
2.0
2.2
2.1
1.9
2.1
2.1
pKa
N
9.1
9.3
9.2
9.4
9.1
9.0
9.3
9.9
9.5
9.8
9.9
9.7
9.7
9.8
10.6
9.2
9.1
10.7
9.3
8.7
pKa
R
8.4
10.5
10.5
12.5
6.0
3.9
4.1 pI
5.65
5.75
5.70
5.95
9.80
10.75
7.65
2.95
3.10
6.10
6.15
6.00
6.00
6.05
6.30
5.70
5.60
5.15
5.70
5.40

http://www.youtube.com/watch?v=w-ctkPUUpUc http://www.youtube.com/watch?v=iaHHgEoa2c8&feature=related

- amino acid linear sequence
- regions of regularly repeating conformations of the peptide chain, such as a
and b
- describes the shape of the fully folded polypeptide chain
- arrangement of two or more polypeptide chains into multisubunit molecule

Primary Structure
Since these side chains stick out from the protein backbone of the molecule, they help determine the properties of the protein made from them
.
amino acid sequence of a protein determines the higher levels of structure of the molecule

The peptide unit (OCNH) is rigid and planar.
The peptide bond (-C-N-) is not free to rotate because it has partial double bond character (resonance).
The peptide bond is 1.32 Å long, between a C-N single bond (1.49 Å) and a
C=N double bond (1.27 Å).
The rigidity of the peptide bond enables proteins to have well-defined 3-D structures.
d
-
..
d
+
Will the peptide C= O be a H-bond donor or acceptor?
Will the peptide NH be a H-bond donor or acceptor?
acceptor donor

Primary Structure
1 mlsifkpalh karlpaaei d ptyrrlrwqi flgiffgyaa yylvrknfal ampylveq gf
61 srg dlgfals gisiaygfsk fimgsvsd rs npr vflpagl ilaaavmlfm gf vpwatssi
121 avmfvllflc gwfqgmgwps cgrtmvh wws qk erggivsv wncahnvggg ippllfllgm
181 awfndwkaa l ympafgaivv alfafam mrd tpqscglppi eeykndypdd ynekaeeelt
241 akqifmqyvl pn kllwyiai anvfvyllry gildwsptyl ke vkhfa ldk sswayflyey
301 agipgtllcg wmsdkvf rgn r gasgvffmt lvtiativyw m npagn pnvd macmiiigfl
361 iygpvmligl hale lapkk a agtaagftgl fgylggsvaa saivgytvd f fgwd ggfmvm
421 iggsilavil lvvvmigekr hhdelqlk rn gg
Ile 421 or I421
Leu189 L189

each protein has its own, unique 3-D shape or native conformation side chains determine the native conformation of a protein
“ likes ” attract: all the hydrophobic side chains (by yellow beads) try to “ get together ” in the center of the molecule, away from the watery environment, hydrophilic side chains are attracted to the outside of the molecule, near the watery environment.

Secondary Structure hydrophilic side chains: acidic OR basic.
In this case “ opposites attract ” acidic side chains with acidic ends are attracted to basic side chains the side chains interacting with each other help to hold the protein in its native conformation
OR water takes the hydrophilic side chains to the “ outside ” of protein

Alpha Helix: the first structure described by Linus Pauling. It has a rod shape.

Not “ correct ” but attempt to show the spiral

Each residue is related to the next one by a rise of 1.5 Å
(translation) along the helix axis and a rotation of 100 o .
Each turn of the helix consists of
3.6 residues, with a pitch of 5.4
Å.
C-terminus
N-terminus http://www.wiley.com/college/fob/quiz/quiz06/6-8.html

N H
CO
Cterminus
N-terminus

Consider the following peptide:
VICKISWACK
The =O of K will be bound to the N-H of ___A____________

Alpha helix secondary structure is represents by a helix.

b
b
b
- polypeptide chains that are almost fully extended
• b
- multiple b strands arranged side-by-side
• b
Strands are stabilized by hydrogen bonds between C=O and -NH on adjacent strands

Irving Geis
..before computer graphics were used to look at biological molecules, an artist
(Irving Geis) (1908-1997) was used.
His renowned paintings, sketches and drawings included cytochromes, viruses, and enzymes..
Most noted: Scientific American , which in
1961 published his painting of myoglobin, the first such depiction of protein crystal structure.
(Article by John Kendrew.)

The polypeptide backbone in a b
-sheet is almost fully extended , rather than being coiled like in a a
-helix.
Pitch
= 7 Å
The NH and CO of each amino acid in one b
-strand are hydrogen bonded to
CO and NH groups of a partner amino acid on another b
-strand to form a b
sheet.

Parallel: parallel with distorted interstrand H-bonds (less stable, bent H-bonds) http://www.wiley.com/college/fob/quiz/quiz06/6-9.html
Antiparallel: antiparallel with linear interstrand Hbonds (more stable)

Beta sheets: some of the strands are parallel and some are antiparallel.
.

Beta sheet secondary structure is represents by an arrow.

H-bonding

•Loops and turns connect a helices and b strands and allow a peptide chain to fold back on itself to make a compact structure
- often contain hydrophilic residues and are found on protein surfaces
- loops containing 5 residues or less
• b
- connect different antiparallel b strands

Random coil: the areas in between
Usually lue and pro

Tertiary Structure

Tertiary Structure tertiary structure or native conformation is a description of the way the whole chain (including the secondary structures) folds itself into its final 3-D shape.
Amino acids which are very distant in the primary structure might be close in the tertiary one because of the folding of the chain !!

Tertiary Structure peptide chain v
H-bonding to form ahelix and bsheet fold on itself native protein

Tertiary Structure
What holds a protein into its tertiary structure?
The tertiary structure of a protein is held together by interactions between the "R" groups.
Electrostatic interactions
Ex:
Amino acids N & E : COO -
Amino acid K : NH
3
+ ionic interaction an electrostatic interaction can occur between the negative and the positive group if the chains folded in such a way that they were close to each other.

Tertiary Structure
Hydrogen bonds hydrogen bonds between side groups - not between groups actually in the backbone of the chain (that governs secondary structure)
Many amino acids contain groups in the side chains which have a hydrogen atom attached to either an O or N.
Ex.
amino acid S -OH group in the side chain hydrogen bond set up between two serine residues in different parts of a folded chain

Tertiary Structure van der Waals (induced dipole:induced dipole)
Many amino acids have quite large hydrocarbon groups in their side chains
Temporary fluctuating dipoles in one of these groups could induce opposite dipoles in another group on a nearby folded chain.
The dispersion forces set up would be enough to hold the folded structure together.

Tertiary Structure
Disulfide Bridges two C side chains end up next to each other because of folding in the peptide chain
S-----S covalent link and so some people count it as a part of the primary structure of the protein.

Tertiary Structure
Metal Ions
A charged metal ion can coordinate to charged side chains to create a fold!
In many instances, the metal is important for enzyme catalysis.

forces that stabilize tertiary structure in proteins

Type structures of globular proteins.

motifs refers to a cluster or grouping of secondary structural units present in many proteins, often serving a similar function.
Top: bab
-motif allow parallel b
-sheets
Middle: helix-turn-helix motif is often found in DNA binding proteins
Bottom: many proteins that bind nucleotides (NAD + ) have a functional motif called a babab
-unit that forms a nucleotide binding site ( Rossmann fold)
42

Domains = stable, structurally independent, globular units which are parts of large proteins (>200 residues) hinge region
Domain 1 Domain 2
Domains have characteristics of small globular proteins (compact, highly folded, soluble in water) often with a specific function for each domain.
43

domains
PDB 1pkn
A domain is a part of protein sequence and structure that exists independently.
Domains are often independently stable.

Quaternary Structure
The quaternary structure is the arrangement of polypeptide subunits within complex proteins made up of two or more subunits v

http://www.youtube.com/watch?v=1eSwDKZQpok&feature=related http://www.youtube.com/watch?v=meNEUTn9Atg&feature=related

protein folding process by which a polypeptide chain acquires its correct 3D structure to achieve the biologically active native state some polypeptide chains spontaneously fold some require the assistance of chaperones : a class of proteins called binds to a partly folded polypeptide chain and prevents it from making associations with other folded or partly folded proteins.
After a polypeptide has acquired most if its correct 2 0 structure, with the
α-helices and β-sheets formed, it has a looser 3 0 structure than the native state and is said to be in the molten globular state.

Proteins are unstable….
X-ray and NMR of proteins tell us that there is one fold with substrates of minor differences between them.
A family of structures that are all ‘ correct ’ .

Proteins are unstable….
So the folding process must be directed thru a kinetic pathway of semi stable intermediates to escape this sampling of irrelevant conformations.

We assume that there are three conformations for each amino acid (ex.
α-helix, β-sheet and random coil). If a protein is made up of 100 amino acid residues, a total number of conformations is
3 100 = 515377520732011331036461129765621272702107522001
= 5 x 10 47 .
If 100 psec (10 -10 sec) were required to convert from a conformation to another one, a random search of all conformations would require
5 x 10 47 x 10 -10 sec = 1.6 x 10 30 years.
However, folding of proteins takes place in msec to sec order. Therefore, proteins fold not via a random search but a more sophisticated search process.

1st observable event in the folding pathway of some proteins is a collapse of the flexible disordered unfolded polypeptide chain into a molten globule.
This event is fast (10 -10 seconds).
Therefore, we (biochemists) know almost nothing about the process!!!
The molten globule has most of the 2 0 structure of the native state but the proper packing interactions have not been formed. http://www.wiley.com/college/boyer/0470003790/animations/protein_folding/protein_folding.htm

Hydrophobic interactions are the driving force for molten globule formation
Next step (up to 1 second), native 3 o structure develops
IMF’s are the driving force to go from molten globule to native (folded) confirmation

Proteins are unstable!!!
changes in pH and temperature can convert protein molecules from native state to a denatured state .
But………….
The energy difference between the native state denatured state in physiological conditions is small, about 5 – 15 kJ/mol ,
P.S. (The energy contribution of a single H-bond is ~2 – 5 kJ/mol.)

What? Did Dr. Mariani just say the energy difference (
D
G) between this: and this:
Is only about 5 – 15 kJ/mol , which is 3X ’ s as strong as a single H-bond
(~2
– 5kJ/mol) ????????

Folding of a polypeptide chain is a thermodynamically favored process.
D
G =
D
H - T
D
S
Internal Interactions - energetically favorable interactions between groups within the folded molecule release heat . These interactions, which include charge-charge, internal hydrogen bonding, and van der
Waals interactions, are the major source of the negative
D
H of folding.
Conformational Entropy - production of a single folded molecule from a multitude of random-coil conformations involves a decrease in randomness and thus a decrease in entropy or a negative
D
S.
A negative
D
S makes a positive contribution to
D
G. http://www.wiley.com/college/pratt/0471393878/student/review/thermodynamics/7_relationship.html

D
G =
D
H - T
D
S
2nd law of thermodynamics: energy is required to create order.
Anything to do with needing energy is quite annoying, which is why entropy (disorder) is preferred.
Entropy: degeneracy
Proteins in the native state are highly ordered in one main conformation whereas the denatured state is highly disordered, with the protein molecules in many different conformations.

D
G =
D
H - T
D
S
2nd law of thermodynamics: energy is required to create order.
A typical unfolded protein contains 1015 – 1020 protein molecules, each of which will have a unique conformation.
They love this!!!!
So… it would be entropically much more favorable for the protein to be in the disordered denatured state.
The energy difference due to entropy between the native ordered state and the denatured state can also reach several hundred kcal/mol.
(but in the opposite direction to the enthalpy difference)

D
S = +
+
HOH
HOH
More Hydrocarbon-Water
Interfacial Area,
More Water Ordered
Less Hydrocarbon-Water
Interfacial Area,
Less Water Ordered interactions between hydrophobic regions of a protein will actually increase entropy by destroying the ordered structures of water around these residues in the unfolded state.

Instability of the native state is biologically very important.
Living cells need globular proteins in correct quantities at appropriate times. It is therefore as important to be able easily to degrade these proteins as it is to be able to synthesize them.
The catalytic activities of enzymes generally require some structural flexibility which would be inconsistent with a completely rigidly stabilized structure.
Proteins are on the brink of instability because easy to move and change.

Proteins are unstable….
A family of structures that are all ‘ correct ’ .
Unstability of the native state is biologically very important.
Proteins are on the brink of instability because easy to move and change.

Often conformational changes play an important role for the function of the protein
Estrogen receptor
With activator (agonist) bound: active
With in-activator (antagonist) bound: not active active inactive

Prion proteins are found in the brains
Function unknown
Two forms normal a
-structure harmful b
-structure b
-structure can aggregate and form
‘plaques’
Blocks certain tissues and functions in the brains

http://www.nsf.gov/news/news_summ.jsp?cntn_id=100689
Protein Data bank
Proteins Nucleic Acids Protein/NA complexes Other Total
X-ray diffraction
NMR
Electron microscopy
Other
Total
32197
5168
95
77
37537
938
735
10
4
1687
1509
125
37
3
1674
28 34672
7 6035
0 142
0 84
35 40933
1MBN
1KCW

Transporter proteins: mediate membrane transport major facilitator superfamily (MFS): typical MFS protein is 400 to 600 amino acids long and has about 12 alpha helixes
GlpT protein: four green helices are located peripherally; three yellow helices line the central pore; four purple helices are located centrally

nonpolar: L A V I F W basic: K R polar (no charge): T C G M N Q Y S acidic: D E (H) on a turn: P and smalls A G V

1 mlsifkpalh karlpaaei d ptyrrlrwqi flgiffgyaa yylvrknfal ampylveq gf
61 srg dlgfals gisiaygfsk fimgsvsd rs npr vflpagl ilaaavmlfm gf vpwatssi
121 avmfvllflc gwfqgmgwps cgrtmvh wws qk erggivsv wncahnvggg ippllfllgm
181 awfndwkaa l ympafgaivv alfafam mrd tpqscglppi eeykndypdd ynekaeeelt
241 akqifmqyvl pn kllwyiai anvfvyllry gildwsptyl ke vkhfa ldk sswayflyey
301 agipgtllcg wmsdkvf rgn r gasgvffmt lvtiativyw m npagn pnvd macmiiigfl
361 iygpvmligl hale lapkk a agtaagftgl fgylggsvaa saivgytvd f fgwd ggfmvm
421 iggsilavil lvvvmigekr hhdelqlk rn gg

1 dptyrrlrwqi flgiffgyaa yylvrknfal ampylveq 7 kllwyiai anvfvyllry gildwsptyl ke
2 dlgfals gisiaygfsk fimgsvsd 8 ldk sswayflyey agipgtllcg wmsdkvf
3
4
5
6 vflpagl ilaaavmlfm avmfvllflc gwfqgmgwps cgrtmvh erggivsv wncahnvggg ippllfllgm lympafgaivv alfafam
9 gasgvffmt lvtiativyw m
10 pnvd macmiiigfl iygpvmligl hale
11 agtaagftgl fgylggsvaa saivgytvd
12 ggfmvm iggsilavil lvvvmigekr hhdelqlk