Download Amino Acids

UNIT I:
Protein Structure and Function
Structure of Proteins
I. Overview
• The 20 common aa’s joined by peptide bonds
• Linear sequence of linked aa’s  info. to generate a
protein molecule with a unique 3D
• Complexity of protein molecules analyzed in terms of 4
organizational levels: 1º, 2º, 3º and 4º
• Analyses revealed that certain structural elements are
repeated in various proteins suggesting general rules
followed in protein folding
• Repeated structural elements range from simple αhelices and β-sheets forming small motifs to complex
folding of polypeptide domains of multifunctional proteins
Figure 2.1. Four hierarchies of protein structure.
II. Primary structure of proteins
• The sequence of aa’s in a protein is called
1º structure of protein
• Many genetic diseases result in proteins
with abnormal aa sequences, causing
improper folding and loss or impairment in
function
• If normal and mutant aa sequences
known, info. may be used to diagnose or
study disease
A. Peptide bond
• In proteins aa’s are
joined covalently by
peptide bonds, i.e.,
amide linkages b/w αcarboxyl of one aa and
α-amino group of
another. e.g.,
valylalanine.
Figure 2.2-A. Formation of a peptide bond, showing
the structure of the dipeptide valylalanine.
• Peptide bonds are not broken by conditions that
denature proteins, e.g., heating or high conc. of
urea.
• Prolonged exposure to a strong acid or base +
elevated temp. is required to hydrolyze nonenzymatically.
1. Naming peptide: the free amino end of
peptide chain (N-terminal) is written to left
and free carboxyl end (C-terminal) to the
right.
•
•
•
Linkage of many aa’s through peptide bonds
 unbranched chain (= polypeptide)
Each aa in a polypeptide is called a “residue”
or “moiety”
When named, aa residues have their suffixes
(-ine, -an, -ic, or –ate) changed to –yl, except
C-terminal aa, e.g., a tripeptide of N-terminal
Val, Gly, C-terminal Leu is: valylglycylleucine.
2. Characteristics of the peptide bond: It has a
partial double-bond character, i.e., it is
shorter than a single bond, and is rigid and
planar.
• This prevents free rotation around the bond b/w the
carbonyl carbon and the nitrogen of the peptide bond
• The bonds b/w the α-carbons and α-amino or αcarboxyl groups can be freely rotated (but limited by
size and character of the R-groups)  allowing
polypeptide chain to assume a variety of possible
configurations
• The peptide bond is generally a trans bond, mainly
because of steric interference of R-groups when in
cis position
Figure 2.2-B. Characteristics of the peptide bond.
3. Polarity of the peptide bond: Like all
amide linkages, the –C=O and –NH
groups of peptide bond are uncharged,
and neither accept nor release protons
over pH 2-12.
•
•
Thus charged groups in a polypeptide
consist of N-terminal α-amino, C-terminal αcarboxyl, and any ionized groups in side
chains.
The –C=O and –NH groups of peptide bond
are polar, and involved in H-bonds, e.g., in
α-helices and β-sheet structures
B. Determination of amino acid composition of a
polypeptide
• First step in determining 1º structure of a polyp. is to
identify and quantitate its aa’s.
• A purified sample of a polyp. to be analyzed is
hydrolyzed by strong acid at 110 ºC for 24 h.
– Treatment cleaves peptide bonds and releases free aa’s 
separated by cation-exchange chromatography.
– The aa’s bind to column with different affinities, depending on
their charges, hydrophobicity and other characteristics
– Each aa is released from column by eluting with solutions of
increasing ionic strength and pH
– Separated aa’s quantitated by heating them with ninhydrin
(forms purple cpd with most aa’s, ammonia, and amide), amount
of each aa is determined spectrophotometrically by measuring
amount of light absorbed by ninhydrin derivatives.
– Analysis described is performed by an automated aa analyzer
Figure 2.3.
Determination of the
amino acid
composition of a
polypeptide using an
amino acid analyzer.
C. Sequencing of the peptide from its N-terminal end
• Sequencing: identifying the specific aa’s at each position
in the polyp. beginning at N-terminal end.
• Phenylisothiocyanate, a.k.a Edman’s reagent, used
to label N-terminal residue under mildly alkaline
conditions.
• Resulting phenylthiohydantoin (PTH) derivative
introduces an instability in N-terminal peptide bond 
selectively hydrolyzed without cleaving other peptide
bonds.
• Identity of aa derivative determined
• Edman’s reagent applied repeatedly to each shortened
polyp. obtained in previous cycle
• Process automated, and repetition is employed by a
machine (sequenator) to determine sequence of > 100
aa’s, starting at N-terminal of a polyp.
D. Cleavage of polypeptide into smaller fragments
•
•
•
Many polyp. composed of > 100 aa’s  cannot be
sequenced directly by a sequenator
Large polyps. cleaved at specific sites 
fragments sequenced
Using more than one cleaving agent (enzymes
and/or chemicals) on separate samples of purified
polyp.  overlapping fragments  permit proper
ordering of sequenced fragments  complete aa
sequence of a large polyp.
Figure 2.5. Overlapping of peptides
produced by the action of trypsin and
cyanogen bromide.
E. Determination of a protein’s primary
structure by DNA sequencing
•
•
•
•
•
Seq. of nucleotides in a coding region of DNA specifies
aa seq. of a polyp.
If nucleotide seq. determined  from genetic code
nucleotide seq. translated  aa seq. of polyp.
This method cannot predict positions of disulfide bonds
in the folded chain
Cannot identify any aa modified after incorporation into
polyp. (post-translational modification)
Direct protein sequencing extremely important tool for
determining true character of 1º sequence of many
polyps.
III. Secondary structure of proteins
• Polyps generally form regular arrangements of
aa’s that are located near each other in the
linear sequence
• These arrangements are called 2º structures of
polyp.
• α-helix, β-sheet, and β-bend are examples of 2º
structures encountered in proteins
• The collagen helix is another example of 2º
structure
A. α-Helix
• Several different polyp. helices, α-helix is most common
• α-helix is a spiral structure, tightly packed, coiled
backbone core, with R-groups extending outward to
avoid interfering sterically with each other.
• Various proteins contain α-helices. e.g., keratins are
fibrous proteins whose structure is nearly entirely αhelical.
– Keratins are a major component of tissues e.g., hair and skin,
and their rigidity is determined by the number of disulfide bonds
b/w constituent polyp chains
• In contrast, myoglobin, with ~80% α-helical, is a globular,
flexible molecule.
1. Hydrogen bonds: An α-helix is stabilized by
hydrogen bonding b/w peptide-bond carbonyl
oxygens and amide hydrogens
–
–
–
The H-bonds extend up the spiral from the carbonyl
oxygen of one peptide bond to the –NH- group of a
peptide linkage 4 residues ahead in the polyp
This ensures that all but first and last peptide bond
components are linked to each other through Hbonds
H-bonds are individually weak, but collectively serve
to stabilize the helix.
Figure 2.6. α-Helix showing
peptide backbone.
2. Amino acids per turn:
– Each turn contains 3.6 aa’s. Thus, aa’s
spaced 3 or 4 apart are spatially close
together when folded in α-helix.
3. Amino acids that disrupt an α-helix:
•
•
•
Proline disrupts α-helix because its imino group is
not compatible with the right handed spiral of α-helix.
Instead it insert a kink in the chain, which interferes
with helical structure
Large number of charged aa’s (e.g., Glu, Asp, His,
Lys, Arg) also disrupt α-helix by forming ionic bonds,
or by electrostatically repelling each other
Amino acids with bulky side chains, e.g., Trp, or aa’s
e.g., Val, Ile, that branch at the β-carbon can interfere
with formation of α-helix if present in large numbers
B. β-Sheet
–
–
–
All peptide bond components
involved in H-bonding.
Surfaces of β-Sheets appear
“pleated”, hence “β-pleated
sheets”
When illustrated, β-strands
are expressed as broad
arrows
Figure 2.7. A.
Structure of a βsheet.
B. An anti-parallel
β-sheet
with the βstrands
represented as
broad arrows.
1. Comparison of a β-sheet and an α-helix
- β-sheets are composed of two or more peptide chains (βstrands), or segments of polyp chains, which are almost fully
extended
- The H-bonds in β-sheets are perpendicular to the polyp
backbone
2. Parallel and antiparallel sheets
- A β-sheet can be formed from ≥ 2 separate polyp chains or
segments of polyp chains arranged either antiparallel or parallel
- When H-bonds are formed b/w the polyp backbones of separate
polyp chains = interchain bonds
- A β-sheet can be formed by a single polyp chain folding back on
itself, here H-bonds = intrachain bonds
- In globular proteins, β-sheets always have right-handed curl, or
twist, when viewed along the polyp backbone
Figure 2.7. B. An anti-parallel β-sheet with the β-strands represented as
broad arrows. C. A parallel β-sheet formed from a single polypeptide
chain folding back on itself.
C. β-Bends (reverse turns)
•
•
•
•
•
Named so because they often connect successive
strands of antiparallel β-sheets
β-Bends reverse the direction of a polyp chain, helping
it form a compact, globular shape
Usually found on surface of protein molecules & often
include charged residues
Generally composed of 4 aa’s, one of which may be
proline. Gly is also frequently found in β-Bends.
Stabilized by H-bonds and ionic bonds
D. Non-repetitive secondary structures
• About one half of an average globular protein is
organized into repetitive structure
• The remainder is described as having a loop or
coil formation.
• These non-repetitive 2º are not “random”, but
rather less regular than those described above
E. Super-secondary structures (motifs)
• Globular proteins constructed by combining 2º
structural elements (α-helices, β-sheets, nonrepetitive)  core region i.e., interior of
molecule.
• They are connected by loop regions (e.g., βbends) at surface of protein.
• Super-secondary structures are usually
produced by packing side chains from adjacent
2º structural elements close to each other
• Thus, e.g., α-helices and β-sheets that are
adjacent in the amino acid sequence are also
usually adjacent in, folded protein.
Figure 2.8. Some common structural motifs combining α-helices and β-sheets.
The names describe their schematic appearance.
IV. Tertiary structure of globular proteins
• 3º refers both to folding of domains (basic units of structure
and function), and final arrangement of domains in a polyp.
• Structure of globular proteins in aqueous soln is compact,
with a high density (close packing) of atoms in core of
molecule.
• Hydrophobic R-groups buried in interior, hydrophilic
groups generally on surface. All hydrophilic groups (including
components of peptide bonds) located in interior are involved
in H-bonds or electrostatic interactions.
• The α-helix & β-sheet structures provide maximal H-bonding
for peptide bond components within interior of polyps 
eliminating possibility that water molecules may become
bound to these hydrophilic groups and, thus, disrupt integrity
of protein
A. Domains
• Fundamental functional and 3D structural units
of a polyp.
• Polyps > 200 aa’s in length generally  2 or
more domains
• Core of domain is built from combinations of
super-secondary structural elements (motifs)
• Folding of peptide chain within a domain usually
occurs independently of folding in other domains
• So, each domain has characteristics of a small,
compact globular protein that is structurally
independent of other domains in polyp chain
B. Interactions stabilizing tertiary structure
•
Four types of interactions cooperate in stabilizing 3º
structures of globular proteins
Disulfide bonds: covalent linkage formed from –SH of
each 2 cysteine residues to produce cystine residue.
1.
–
–
–
–
The 2 Cys residues may be far apart in the 1º sequence or
even on 2 different polyps.
Folding of polyp chain(s) brings Cys residues into proximity
Contribute to stability of 3D shape of protein e.g., many
disulfide bonds are found in proteins as Ig’s that are secreted
by cells
These strong covalent bonds prevent proteins from becoming
denatured in extracellular environ.
Figure 2.9.
Formation of a disulfide bond by
the oxidation of two cysteine
residues, producing one cystine
residue.
2. Hydrophobic interactions: aa’s with nonpolar R-groups tend to be located in
interior of polyp, where they associate
with other hydrophobic aa’s
– In contrast, polar or charged aa’s tend to be
located on surface in contact with polar
solvent
•
For protein located in non-polar (lipid)
environ. e.g., membrane, aa’s exhibit
reverse arrangement.
Figure 2.10
Hydrophobic interactions
between amino acids with nonpolar side chains.
Figure 1.4
Location of nonpolar amino acids in soluble and
membrane proteins.
3. Hydrogen bonds: aa R-groups containing
oxygen- or nitrogen-bound hydrogen,
e.g. alcohol groups of Ser and Thr, can
form H-bonds with electron rich atoms
e.g., oxygen of a carboxyl group or
carbonyl group of a peptide bond
– Formation of H-bonds b/w polar groups on
protein surface and aqueous solvent
enhances solubility of protein
Figure 2.11
Interactions of side chains of
amino acids through hydrogen
bonds and ionic bonds.
Figure 1.6
Hydrogen bond between the phenolic hydroxyl group of
tyrosine and another molecule containing a carbonyl group.
4. Ionic interactions: negatively charged
groups, e.g., (-COO-) in side chains of
Asp or Glu can interact with positively
charged groups, e.g., (-NH3+) in side
chain of Lys
C. Protein folding
• Interactions b/w side chains of aa’s determine folding of
long polyp into 3D shape of functional protein
• Protein folding, occurs within cell in seconds to minutes,
employs a shortcut through the maze of all folding
possibilities
• As a peptide folds, its aa R groups attracted and
repulsed according to their chemical properties, e.g.,
charged side chains
• Plus, interactions involving H-bonds, hydrophobic
interactions, and disulfide bonds
• This process of trial and error tests many possible
configurations, where interactions outweigh repulsions
 correctly folded protein with a low energy state
Figure 2.12. Steps in protein folding.
D. Role of chaperones in protein folding
• Generally accepted: info needed for correct
protein folding is contained in 1º structure of
polyp.
• Difficult to explain, why most proteins when
denatured do not resume native
conformation
• One answer: a protein begins to fold in
stages during its synthesis, rather than
waiting completion of synthesis of entire
polyp  limits competing folding
configurations made available by longer
stretches of nascent polyp.
• Additionally, a specialized group of
proteins “chaperones” required for proper
folding of many proteins.
– Chaperones, a.k.a. “heat shock” proteins
interact with polyp at various stages during
folding
– Some chaperones are important in keeping
polyp unfolded until its synthesis finished, or
act as catalysts by increasing rates of final
stages in folding process
– Others protect proteins as they fold so that
their vulnerable, exposed regions do not
become tangled in unproductive encounters
V. Quaternary structure of proteins
• Many proteins consist of a single polyp. 
monomeric proteins
• Others may consist of ≥ 2 polyp chains that may
be structurally identical or totally unrelated
• Arrangement of these polyp subunits =
quaternary structure ( 2 subunits = dimeric, 3
subunits = trimeric, several subunits =
multimeric)
• Subunits held together by non-covalent
interactions (H-bonds, ionic, hydrophobic)
• Subunits may function independently of each
other, or cooperate as in Hb  binding of
oxygen to one subunit of the tetramer increases
affinity of the other subunits for oxygen
VI. Denaturation of proteins
• Results in unfolding and disorganization of proteins’ 2º
and 3º structures, not accompanied by hydrolysis of
peptide bonds
• Denaturing agents include heat, organic solvents,
mechanical mixing, strong acids or bases, detergents
and ions of heavy metals e.g., lead and mercury
• Denaturation may under ideal conditions be reversible
i.e., protein refolds into original native structure when
denaturing agent removed
• Most proteins, once denatured, remain permanently
disordered
• Denatured proteins are often insoluble and, so
precipitate in solution
VII. Protein misfolding
• Protein folding is a complex, trial and error
process that sometimes  improperly folded
protein
• Misfolded proteins usually tagged and degraded
within the cell but this quality control system is
not perfect and intracellular or extracellular
aggregates of misfolded proteins can
accumulate, particularly as individuals age
• Deposits of misfolded proteins are associated
with a number of diseases including
amyloidoses
A. Amyloidoses
• Misfolding of proteins may occur spontaneously,
or by mutation in a gene which produces altered
protein
• Plus, some apparently normal proteins 
abnormal proteolytic cleavage  unique
conformational state  long, fibrillar protein
assemblies consisting of β-pleated sheets
• Accumulation of these spontaneously
aggregating proteins, called “amyloides”, has
been implicated in many degenerating diseasesparticularly in the neurodegenerative disorder,
Alzheimer disease.
Alzheimer
• Dominant component of amyloid plaque that
accumulates in Alzheimer is Aβ, a peptide 40-43 aa’s
• X-ray crystallography and IR spectroscopy demonstrate
a characteristic β-pleated sheet conformation in nonbranching fibrils
• This peptide, when aggregated in β-pleated sheet
configuration, is neurotoxic, is central pathogenic event
 cognitive impairment characteristic of disease
• Aβ amyloid deposited in brain in Alzheimer is derived by
proteolytic cleavages from the larger amyloid precursor
protein – a single transmembrane protein expressed on
cell surface in brain and other tissues
• Aβ peptides aggregate  amyloid found in brain
parenchyma and around blood vessels.
Figure 2.13. Formation of amyloid plaques found in Alzheimer disease.
• Most Alzheimer cases are not genetically
based, although 5-10% of cases are
familial
• A second biologic factor involved in
development of Alzheimer is accumulation
of neurofibrillay tangles in brain
– A key component of these tangled fibers is an
abnormal form of tau protein, which in healthy
form  helps in assembly of microtubular
structure
– Defective tau, however, appears to block
actions of its normal counterpart
B. Prion disease
• Prion protein “PrP”, implicated as causative agent of
transmissible spongiform encephalopathies (TSE),
including:
– Creutzfeldt-Jakob disease in humans
– Scrapie in sheep
– Bovine spongiform encephalopathy “BSE” in cattle (a.k.a
“mad cow disease”)
• Infectivity of agent causing scrapie was associated with
a single protein species that was not associated with
detectable nucleic acid
• Infectious protein is designated “prion protein”, is highly
resistant to proteolytic degradation
• When infectious tends to form insoluble aggregates of
fibrils, similar to amyloid found in other brain diseases
• A non-infectious form of PrP, having same aa and gene
sequences as infectious agent, is present in normal
brains on surface of neurons and glial cells
• i.e., PrP is a host protein. No 1º structure differences or
alternate posttranslational modifications b/w normal and
infectious forms
• The key to becoming infectious lies in changes in 3D
conformation of PrP, e.g., a number of α-helices replaced
by β-sheets in infectious form
• Presumably, conformational difference  relative
resistant to proteolytic degradation & permits
distinguishing infectious from normal
• Infectious agent is an altered version of normal protein,
which acts as a “template” for converting normal protein
to pathogenic conformation
• TSEs are invariably fatal, no treatment is available
Figure 2.14.
One proposed mechanism
for multiplication of infectious
prion agents.
Summary
• Native conformation : functional fully-folded protein structure
• 3D structure is determined by 1º structure (i.e., aa sequence)
• Interactions b/w R-groups guide folding  2º, 3º, and sometimes
4º
• A specialized group “Chaperones” required for proper folding of
many proteins
• Protein denaturation  unfolding & disorganization of protein’s
structure without hydrolyzing peptide bonds
• Denaturation may be reversible or, more commonly irreversible
• Disease can occur as a normal protein  a conformation that is
cytotoxic e.g., Alzheimer and TSEs
• In Alzheimer, normal protein after abnormal processing 
conformation  neurotoxic amyloid protein consisting of βpleated sheets. Additionally, abnormal tau protein
• In TSEs infective agent is an altered version of a normal prion
protein that acts as a template for converting normal protein to
pathogenic conformation