Review:

1

• Review
• Gibbs free energy, enthalpy, entropy
• Work and energy storage/utilization in biological
  systems
• Types of chemical bonding (covalent, H-bonding,
  electrostatic, Van der Waals)
• The hydrophobic effect
• Protein microenvironments
• Properties of water, acid-base equilibria

2
GMS BI 555/755 Lecture 2 Levels of Protein
Structure

• Reading Berg 6th ed. Chapter 2 (Supplemental
  Creighton Proteins)
• Primary Structure
• Amino acid side chains and classification
• The peptide bond
• Secondary structure
• Peptide bond angles and rotation
• Alpha helix
• Beta sheet
• Turns
• Tertiary structure
• Hydrophobic effect
• Effects of solvent
• Folding motifs
• Protein folding problem
• Molecular chaperones
• Quaternary structure

3
Protein primary structure
Proteins are polymers of L-amino acids linked by
peptide bonds
Amide bond
60
40

• Amide bonds have a substantial degree of planar
  character
• Chemically unreactive. Hydrolysis at pH extremes

4
The peptide bond

• Peptide (amide) bond very stable in solution in
  the absence of a catalyst

Peptide bonds may be trans or cis, trans being
favored because there are fewer unfavorable
steric interactions
5
Formation of the peptide bond

• Peptide bond is an amide bond
• Very stable
• Positive ?H
• Peptide bond formation increases order (negative
  ?S)
• Not a spontaneous process (becomes spontaneous
  when coupled to a process such as ATP hydrolosys)

6
Glycine and alanine

• A, G
• Neutral
• Small R group (low accessible surface area)
• Non-polar
• Flexible
• G achiral, most flexible AA

7
Aliphatic amino acids

• V,L,I,M
• Neutral
• High surface area
• Non-polar
• Hydrophobic
• Van der Waals interactions in folded interior
• Structural units with a variety of shapes
• I side chain is chiral
• V,L,I common
• M Rare
• Easily oxidized to sulfoxide then sulfone

8
Methionine oxidation
Methionine residues are susceptible to oxidation
in vivo and during protein workup and
characterization
9
Aromatic amino acids
A eBC A absorbance e molar absorbtivity C
concentration

• F, Y, W
• Neutral
• Very high accessible surface area
• F very non-polar, hydrophobic
• W rare
• W, Y responsible for 280 nm absorbance
• W Fluorescent properties

10
AAs with alphatic hydroxyl group

• S, T
• Neutral
• Polar, H-bonding donors or acceptors
• Hydrophilic or hydrophobic
• Sites of post-translational modification
• Phosphorylation (S, T, Y)
• O-glycosylation ( ß-O-GlcNAc, O-glycans)
• T side chain chiral

11
Cysteine

• Sulfhydryl (thiol) most reactive group in
  proteins
• Oxidation in presence of oxygen
• Very nucleophilic, reactions with electrophiles
• Must be alkylated (stabilized) for effective
  analysis
• Reactions with metal ions
• Participates in disulfide bonding with other
  cysteine residues. Important secondary structure
  stabilizing event in proteins.
• Antioxidant, precursor to glutathione

Cys is nucleophilic and must be alkylated for
analysis (reaction with iodoacetic acid)
Cystine disulfide bonded Cys residues
12
Cysteine alkylation
Fluorescent alkyl groups
Derivatizing groups for cys stabilization
Fluoresceine-5-maleimide
13
Homocysteine analog of Cys and Met, metabolic
intermediate

• Elevations of homocysteine occur in the rare
  hereditary disease homocystinuria and in the
  methylene-tetrahydrofolate-reductase polymorphism
  genetic traits. The latter is quite common (about
  10 of the world population) and it is linked to
  an increased incidence of thrombosis and
  cardiovascular disease and that occurs more often
  in people with above minimal levels of
  homocysteine (about 6 µmol/L)
• Risk factor for vascular disease, Alzheimers
  Disease

Darvesh, S., Walsh, R., and Martin, E. (2007)
Homocysteine Thiolactone and Human
Cholinesterases. Cell Mol Neurobiol 27, 33-48.
14
The basic amino acids

• R, K
• Positively charged at pH 7
• Most basic protein groups (also N-term)
• H
• Can participate in acid/base reactions at pH 7

pKa 6
pKa 12
nucleophilic
Histidine Ionization. Histidine can bind or
release protons near physiological pH.
15
Hydroxylysine

• Biosynthesized from lysine oxidation by lysyl
  oxidase
• Found only in animal proteins, mostly in collagen
  as a PTM
• 6-67 of 1000 AA residues of collagen are
  hydroxylysine
• 17-90 of collagen lysyl residues are
  hydroxylated
• Hydroxylysine is typically found in
  triple-helical regions almost exclusively in the
  Y positions of the repeating -X-Y-Gly- sequences
  in various collagens.
• Embryonic tissues contain much more hydroxylysine
  than adult tissues.
• Hydroxylation of lysyl residues in collagens
  prevents deposit of minerals between fibrils
• Lysine hydroxylation seems to be increased as
  well in some diseases, for example,
  lipodermatosclerosis, osteoporosis, and
  osteogenesis imperfecta
• Precursor to collagen crosslinking
• Hydroxylated lys residues may be glycosylated

http//herkules.oulu.fi/isbn9514267990/html/x319.h
tml
16
Ornithine (analog of Lys, product of arginase)
Ornithine lactamization ornithine is unstable in
peptide chains due to its propensity to form
6-membered cyclic lactams
Ornithine is one of the products of the action of
the enzyme arginase on L-arginine, creating urea.
Therefore, ornithine is a central part of the
urea cycle, which allows for the disposal of
excess nitrogen. Ornithine is not an amino acid
coded for by DNA, and, in that sense, is not
involved in protein synthesis. However, in
mammalian non-hepatic tissues, the main use of
the urea cycle is in arginine biosynthesis, so as
an intermediate in metabolic processes, ornithine
is quite important (wikipedia)
17
AAs with side chain carbonyls

• N,Q
• Neutral
• Polar, H-bonding
• Deamidation reactions (protein ageing)

• D,E
• pKa5
• Very polar
• Usually charged in proteins
• Esterification reactions possible

D, E are acidic, hydrophilic
Neutral, hydrophilic
18
Protein deamidation

• Deamidation is a common post-translational
  modification
• Conversion of Asn to a mixture of Asp and
  isoaspartate (aka beta-aspartate).
• Occurs to a lesser extent with Gln
• Deamidation may cause loss of protein activity
• An important consideration for recombinant
  protein-based drugs and therapeutics
• Occurs in vivo, especially among proteins with
  long life times.
• Highest frequency for Asn-Gly sequences
• Intermediate frequency for Asn-X where X polar
  (Ser, Thr, Asp)
• Low frequency for Asn-X where X hydrophobic
  residue
• Asn must be on flexible portion of protein
• Alkaline pH accelerates deamidation
• Change in protein acidity

19
Proline

• Cyclic imino acid
• No rotaton about N-Ca bond
• No backbone N-H H-bonding.
• No resonance stabilization of amide bond
• Peptide bond more likely to be in
  cis-conformation

Trans and Cis X-Pro Bonds. The energies of these
forms are relatively balanced because steric
clashes occur in both forms.
20
Hydroxyproline
There are 28 types of collagen, over 90 of the
collagen in the body are of type I, II, III, and
IV. Collagen One - bone (main component of bone)
Collagen Two - cartilage (main component of
cartilage) Collagen Three - reticulate (main
component of reticular fibers) Collagen Four -
floor key component of basement membranes
Twisted, left handed helix due to high Pro, Gly
content.

• Hydroxyproline is produced by hydroxylation of
  the amino acid proline by the enzyme prolyl
  hydroxylase following protein synthesis (as a
  post-translational modification). The enzyme
  catalysed reaction takes place in the lumen of
  the endoplasmic reticulum.
• Hydroxyproline is a major component of the
  protein collagen.
• Hydroxyproline and proline play key roles for
  collagen stability. They permit the sharp
  twisting of the collagen helix. In the canonical
  collagen Xaa-Yaa-Gly triad (where Xaa and Yaa are
  any amino acid), a proline occupying the Yaa
  position is hydroxylated to give a Xaa-Hyp-Gly
  sequence. This modification of the proline
  residue increases the stability of the collagen
  triple helix.
• It was initially proposed that the stabilization
  was due to water molecules forming a hydrogen
  bonding network linking the prolyl hydroxyl
  groups and the main-chain carbonyl groups. It
  was subsequently shown that the increase in
  stability is primarily through stereoelectronic
  effects and that hydration of the hydroxyproline
  residues provides little or no additional
  stability.
• Hydroxyproline is found in few (animal) proteins
  other than collagen. The only other mammalian
  protein which includes hydroxyproline is elastin.
  For this reason, hydroxyproline content has been
  used as an indicator to determine collagen and/or
  gelatin amount. (wikipedia)

21
Space filling amino acid side chain structures
Lesk Introduction to Protein Science, chap 3,
Fig. 1
22
(No Transcript)
23
Codon usage and protein structure evolution
24
Table of the frequency with which one amino acid
is replaced by others in the amino acid sequence
of the same protein in different organisms
25
Rotation of peptide bonds in a polypeptide
Dihedral angles in a polypeptide
Rotation About Bonds in a Polypeptide. The
structure of each amino acid in a polypeptide can
be adjusted by rotation about two single bonds.
(A) Phi (f) is the angle of rotation about the
bond between the nitrogen and the a-carbon atoms,
whereas psi (y) is the angle of rotation about
the bond between the a-carbon and the carbonyl
carbon atoms. (B) A view down the bond between
the nitrogen and the a-carbon atoms, showing how
f is measured. (C) A view down the bond between
the a-carbon and the carbonyl carbon atoms,
showing how y is measured.
The dihedral angles of a sequence of amino acid
residues defines the three dimensional structure
of the protein backbone
A Ramachandran Diagram Showing the Values of f
and ?. Not all f and ? values are possible
without collisions between atoms. The most
favorable regions are shown in dark green
borderline regions are shown in light green. The
structure on the right is disfavored because of
steric clashes.
26
Protein secondary structure alpha helix

• Structure of the a Helix. (A) A ribbon depiction
  with the a-carbon atoms and side chains (green)
  shown. (B) A side view of a ball-and-stick
  version depicts the hydrogen bonds (dashed lines)
  between NH and CO groups. (C) An end view shows
  the coiled backbone as the inside of the helix
  and the side chains (green) projecting outward.
  (D) A space-filling view of part C shows the
  tightly packed interior core of the helix.
• 3.6 res/turn
• H-bonding to i4

27
(No Transcript)
28
Proteins with high a-helical content
A Largely a Helical Protein.      Ferritin, an
iron-storage protein, is built from a bundle of a
helices.
Myoglobin first protein structure reconstructed
by X-ray crystallography (Kendrew and Perutz),
proved prediction of a-helix structure by Corey
and Pauling
29
Protein secondary structure ß-sheets
An Antiparallel ß Sheet. Adjacent ß strands run
in opposite directions. Hydrogen bonds between NH
and CO groups connect each amino acid to a single
amino acid on an adjacent strand, stabilizing the
structure.
A Parallel ß Sheet. Adjacent ß strands run in the
same direction. Hydrogen bonds connect each amino
acid on one strand with two different amino acids
on the adjacent strand.
30
Protein secondary structure ß-sheets
Ribbon diagrams of twisted ß-sheets
A mixed ß-sheet
31
Protein secondary structure ß-sheets
Structure of a Reverse Turn. The CO group of
residue i of the polypeptide chain is hydrogen
bonded to the NH group of residue i 3 to
stabilize the turn
Loops on a Protein Surface. A part of an
antibody molecule has surface loops (shown in
red) that mediate interactions with other
molecules
32

• What determines whether a particular protein
  sequence (sub-sequence) forms an a-helix,
  ß-sheet, or a turn?
• Amino acid residues have varying propensities to
  be present in secondary structures.
• a-helix (default), branched R-groups disfavored
  (V,T,I) H-bond donating R-groups disfavored (S,
  D, N)
• ß-strands more tolerant of bulky R groups
• Proline disrupts a-helices and ß-sheets, found in
  turns.

Values difft in 5th ed
33
Hydropathicity/hydrophobicity index
KD Hydrophobicity plot for human rhodopsin
Kyte and Doolittle hydrophobicity
Expasy (http//ca.expasy.org)
Kyte, J., and Doolittle, R. F. (1982) J Mol Biol
157, 105-132.
34
Prediction of protein secondary structure from AA
sequence
KD
Chou and Fasman
Computed scale of alpha helix forming properties
for the 20 AA based on known protein structures
Chou P.Y., Fasman G.D. Adv. Enzym.
4745-148(1978).
35
Tertiary structure the overall three dimensional
fold of a polypeptide chain
Ball and stick model showing all myoglobin atoms
but not showing the amount of space each occupies
Diagram depicting the amino acid backbone of
myoglobin as a ribbon (8 helices) but no side
chains
36
Tertiary structure of proteins driven by the
hydrophobic effect
Distribution of Amino Acids in Myoglobin. (A) A
space-filling model of myoglobin with hydrophobic
amino acids shown in yellow, charged amino acids
shown in blue, and others shown in white. The
surface of the molecule has many charged amino
acids, as well as some hydrophobic amino acids.
(B) A cross-sectional view shows that mostly
hydrophobic amino acids are found on the inside
of the structure, whereas the charged amino acids
are found on the protein surface.
Figure 3.46. Inside Out Amino Acid Distribution
in Porin. The outside of porin (which contacts
hydrophobic groups in membranes) is covered
largely with hydrophobic residues, whereas the
center includes a water-filled channel lined with
charged and polar amino acids.
37
Protein sequence motifs structural elements
(folds) found in different proteins

• Protein motifs are three dimensional structures
  (folds) found in a diversity of proteins and
  protein families. Their presence may imply a
  certain class of function (structural, enzymatic,
  or adhesive)
• Algorithms exist for predicting the presence of
  motifs from the primary sequence.

Greek key motif of beta strands
38
Protein folding motifs
Richardson, J. S. (1994) Introduction protein
motifs. Faseb J 8, 1237-9.
39
The role of solvent in secondary/tertiary
structure formation
Reduction and denaturation of ribonuclease

• Chaotropic (denaturing) agents form H-bonds with
  water, disrupt the normal structure of water,
  change the energetic balance that favors
  sequestering hydrophobic sequences in interior
  domains. In the absence of the entropic driving
  force behind protein folding, unfolding occurs.

Anfinson, 1950s Reduced, denatured ribonuclease
regains enzymatic activity when urea and
ß-mecaptoethanol are removed by dialysis
SDS
40
Protein folding

• Proteins have the capacity to fold and become
  active based on the information contained in
  their amino acid sequence.
• Thermodynamically spontaneous
• Proteins fold in buffered water
• Chaotropic agents disrupt the structure of water
  by participating in hydrogen bonding. As a
  result, the hydrophobic driving force that makes
  a folded structure energetically favorable is
  disrupted
• Guanidine salts, urea, detergents
• Proteins also denature at pH values deviating
  significantly from neutral.
• Water miscible organic solvents are able to
  participate in hydrogen bonding. Their presence
  also alters the thermodynamic driving force
  behind protein folding. As the percent of
  organic solvent in a solution increases, the
  tendency of proteins to unfold increases.
• Heat increases molecular motion. As proteins
  heat they fold and unfold rapidly.
  Intermolecular interactions of hydrophobic
  domains may cause proteins to precipitate
  (cooking an egg).

41
Proteins fold cooperatively, not randomly
A current view of protein folding. Each domain of
a newly synthesized protein rapidly attains a
molten globule state. Subsequent folding occurs
more slowly and by multiple pathways, often
involving the help of a molecular chaperone. Some
molecules may still fail to fold correctly.
These are recognized and degraded by specific
proteases.
Components of a Partially Denatured Protein
Solution. In a half-unfolded protein solution,
half the molecules are fully folded and half are
fully unfolded. (Berg). There are too many
possible structures for a random process
(Levinthals paradox, 5 x 1047 structures for 100
aa protein). Progressive stabilization of
intermediates results in correctly folded
proteins.
Lodish
42
(No Transcript)
43
Molecular chaperones stabilize hydrophobic
sequences of newly synthesized polypeptides to
enable orderly folding

• Improperly folded proteins do not exit the ER
• HSPs heat shock proteins, so named because their
  expression increases in response to heat and
  other cellular stresses that result in buildup of
  mis-folded proteins
• HSPs require energy.
• Polypeptides carry the information to fold in
  their sequences with assistance from chaperones

44
The GroEL/GroES (Hsp60/Hsp10) chaperone machine
Richardson, A., Landry, S. J., and Georgopoulos,
C. (1998) Trends Biochem Sci 23, 138-43.
45
Molecular chaperones and protein folding quality
control example calnexin and protein
N-glycosylation
The role of N-linked glycosylation in ER protein
folding. The ER-membrane-bound chaperone protein
calnexin binds to incompletely folded proteins
containing one terminal glucose on N-linked
oligosaccharides, trapping the protein in the ER.
Removal of the terminal glucose by a glucosidase
releases the protein from calnexin. A glucosyl
transferase is the crucial enzyme that determines
whether the protein is folded properly or not if
the protein is still incompletely folded, the
enzyme transfers a new glucose from UDP-glucose
to the N-linked oligosaccharide, renewing the
protein's affinity for calnexin and retaining it
in the ER. The cycle repeats until the protein
has folded completely. Calreticulin functions
similarly, except that it is a soluble ER
resident protein. Another ER chaperone, ERp57
(not shown), collaborates with calnexin and
calreticulin in retaining an incompletely folded
protein in the ER.
Lodish
46
The protein folding problem can we predict the
three dimensional structure of a protein from its
amino acid sequence

• The sequence contains the information necessary
  for folding
• Useful predictions of secondary structure can be
  made (numerous tools on web, Expasy)
• A given peptide sequence may produce more than
  one fold in different proteins
• Conformational preferences of AAs not absolute
• Tertiary interactions among residues far apart in
  sequence influence the formation of secondary
  structure.
• The integration of secondary structures is a very
  computationally intensive problem.
• There is steady progress in understanding
  polypeptide properties but no clear solution to
  the protein folding problem (Nobel Prize!)

47

• Free energy change of protein folding
• Unfolded proteins are random, folding entails
  considerable increase in order (so, why does it
  occur spontaneously?)
• Water molecules must form highly ordered cages
  around hydrophobic aa residues. Folding shields
  these residues from water, balancing the apparent
  increase in order.
• H-bonding, electrostatic and Van der Waals
  interactions results in a release in heat
  (negative enthalpy, ?H)

48
Lesk, chap 5 Fig. 8
49
Quaternary structure spatial arrangement of
multi subunit proteins (made of more than one
polypeptide chain)
A simple dimer Quaternary Structure. The Cro
protein of bacteriophage ? is a dimer of
identical subunits.
A hetero-tetramer hemoglobin is composed of 2 a
and 2 ß chains, each with a heme group.
Quaternary structure results from numerous
interactions between the surfaces of the protein
molecules. Structural plasticity allows
cooperative oxygen binding in hemoglobin.
Molecular machines are multiprotein complexes
that execute many of the important functions in
the cell (ribosome, nuclear pore complex, etc)
50
Protein quaternary structure
51
Micro-environments and macromolecular complexes
Atomic structure of the 50S Ribosome Subunit.
Proteins are shown in blue and the two RNA
strands in orange and yellow. The small patch of
green in the center of the subunit is the active
site. (Wikipedia)
The eukaryotic membrane, showing lipid bilayer,
integral membrane proteins, protein channel,
glycolipids
52
X-Ray Crystallography

• Steps
• Recombinant expression of protein
• Formation of large, pure crystals, regular in
  structure, no imperfections
• X-ray exposure, measurement of diffraction
  pattern, as crystal is rotated
• Computation on raw data, refinement, model
  building
• Repeat

• 36,000 protein structures solved to date using
  X-ray crystallography
• Crystal formation difficult for membrane proteins
• Very bright X-ray source needed (synchrotron) to
  produce the highest resolution (national labs)

Wikipedia
53
NMR Spectroscopy

• Nuclear magnetic resonance measures the
  environment of protons and other nuclei (13C,
  15N, 31P)
• CH, NH, OH, COOH, etc
• NMR experiments determine distance constraints
  between NMR active nuclei in biomolecules
• High concentration, high purity protein needed
• Able to measure protein dynamics
• Limited ability to solve structures of very large
  proteins
• Expression of isotope enriched proteins to
  maximize NMR sensitivity
• Requires a very large magnet (900 MHz NMR
  spectrometer, above)
• Resource and computationally intensive

6000 protein structures solved
Wuthrich, K. (1990) J Biol Chem 265, 22059-22062.