Download The Three Dimensional Structure of Proteins

The Three Dimensional Structure of Proteins
Objectives:
I. Describe the Native State or Native Conformation of a Protein.
A. Review the difference between Conformation and Configuration.
B. Why is the correct three dimensional structure of a protein essential to its function?
C. Explain the importance of amino acid sequence in protein structure and function.
D. What is protein breathing
II. Describe the types of secondary (2°) structure.
A. Helical structures; the α-helix.
B. Sheet like structures; the β-sheet.
C. Turns or bends
1. type I β-turn vs. type II β-turn.
D. Understand the intramolecular force(s) that maintain secondary (2°) structure.
E. Groups on the peptide / protein involved in stabilizing 2° structure.
III. Describe the tertiary (3°) structure of a protein.
A. Describe the intramolecular forces that stabilize 3° structure.
B. Understand the R group interactions that maintain the three dimensional conformation of a
protein.
IV. Recognize folding patterns within the native folded protein.
A. Describe structural motifs.
B. Describe domains.
1. Differences between motifs and domains.
V. Describe quaternary (4°) structure of a protein.
A. Describe the intramolecular forces that stabilize 4° structure.
B. Understand the R group interactions that maintain the three dimensional conformation of a
protein.
VI. Random Structures??
A. Describe Non-Regular Non-Repeating Structures.
B. Describe Intrinsically Unstructured Proteins or Natively Unfolded Proteins.
1. Differences between Non-Regular Non-Repeating Structures and Intrinsically Unstructured
Proteins
VII. Describe the folding process.
A. The unfavorable Entropy change in the protein molecule as it folds.
1. The protein would favor a random extended structure.
B. The favorable Enthalpy change arising from intramolecular side chain interactions.
C. The favorable Entropy change arising from burying the hydrophobic groups within the molecule
and the concomitant release of structured water - The Hydrophobic Effect.
D. The postulated hierarchy of folding events.
E. The function of chaperones and chaperonins.
1. Differences between chaperones and chaperonins.
VIII. Classification and properties of proteins based upon their final folded form.
A. Fibrous
B. Globular
C. Membrane
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IX. Classification of proteins based upon the presence or absence of a prosthetic group.
A. Conjugated versus Unconjugated proteins.
B. Types of prosthetic groups.
1. Metals
2. Heme
3. Lipids
4. Carbohydrates, etc.
a) O-linked vs. N-linked
C. Apoprotein vs. Holoprotein.
X. Describe on a molecular level the denaturation of a protein.
A. Distinguish between Hydrolysis and Denaturation.
1. Level(s) of structure affected
B. Agents / Actions the denature proteins.
1. Heat
2. Physical agitation
3. pH changes
4. Detergents
5. Organic Solvents
6. Chaotropic agents (urea, guanidinium hydrochloride, etc.)
7. Heavy metals
XI. Be familiar with the structures and functions of the fibrous proteins.
A. Collagen
1. Primary structure.
2. Secondary structure.
3. Tertiary structure.
4. Quaternary structure
5. Understand the role Vitamin C plays in collagen metabolism.
XII. Myoglobin & Hemoglobin as typical globular proteins
XIII. Prion Related Diseases - Transmissible Spongiform Encephalopathies.
A. Diseases of incorrect (abnormal) protein folding.
1. Structural changes.
2. “Functional” changes.
3. How / Why the prion protein takes on the abnormal conformation?
Background / Introduction
The correct primary (1°) structure is not sufficient for a protein to have biological activity. Before a protein
gains its biological activity, the long linear polymer of amino acids must assume a unique three dimensional
shape, it must fold into a precise three dimensional conformation. The conformation attained by the folded
protein is the thermodynamically most favored shape; the energy of the system is at a minimum and the
entropy of the system is at a maximum. This final folded most stable state of the protein is called the
NATIVE STATE or NATIVE CONFORMATION. A multitude of weak intramolecular and in some cases
intermolecular interactions stabilize the native conformation of a protein. These weak intramolecular
interactions have life times in the femtosecond to picosecond range so these stabilizing “bonds” are always
breaking and reforming. Proteins in the cell vacillate from the absolute most stable form to forms slightly
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less favorable and back again to the most favored form. This vacillation around a most stable conformation
is due to the breaking and reforming of the weak interactions and is called PROTEIN BREATHING.
Levels of Protein Structure
Protein Structure has historically been divided into four (4) main levels. As protein structure has been
studied in more detail, several sub-levels of structure have been added.
PRIMARY (1°) STRUCTURE is the unique amino acid sequence of the protein.
SECONDARY (2°) STRUCTURES are regular repeating structures that occur over short localized regions of the
protein molecule. Secondary structure refers to an “isolated” segment of polypeptide chain and describes
the local spatial arrangement of its main chain atoms, without regard to the positioning of its side chains or
its relationship to other segments. They are stabilized by hydrogen bonds between the carbonyl oxygens
and amido hydrogens of the peptide bond.
TERTIARY STRUCTURES (3°) are long range interactions within the protein molecule; regions of the protein
separated by long distances interact with each other. These long range interactions fold the molecule into a
compact shape. Tertiary structures involve contact among and between the amino acid side chains of the
molecule. These structures are stabilized by hydrophobic interactions between hydrophobic side chains, by
polar interactions (dipole-dipole & hydrogen bonding) between polar side chains, by interactions between
polar groups of the peptide bond and polar side chains, and by electrostatic interactions.
QUATERNARY (4°) STRUCTURE is the association of two or more polypeptide chains (protomers / subunits)
into a functional molecule. Quaternary structure involves the formation of a multimeric protein. Protein
molecules composed of two or more subunits by definition have quaternary structure.
Secondary Structures
How then is it possible for a protein to find one conformation or a group of closely
related conformations that are energetically most favorable? More importantly how
and why do certain short localized regions of the protein fold into regular repeating 2°
structures? The answers to these questions lie in the partial double bond nature of the
peptide bond. Rotation around the C–N peptide bond is greatly restricted and the six
atoms of the peptide bond all lie in the same plane which greatly limits the number of
allowable stable conformations.
Carbonyl
Oxygen
O
C
C
C
N
H
Amido
Hydrogen
Much of the early work on protein secondary structures was performed by two groups.
One group, headed by G.N. Ramachandran calculated the permitted conformations of a
polypeptide chain. The calculations were based upon allowed bond rotations along the peptide backbone,
Van der Waals radii of the backbone atoms, and X-ray crystallographic data. The second group, Linus
Pauling and Robert Cory, performed X-ray crystallographic studies on simple peptides and built models to
fit the data. Both groups arrived at the same conclusions: there are a limited number allowed
conformations; a limited number of 2° structures. The allowed 2° structures include a circular structure that
has never been observed in naturally occurring proteins, helical structures (right handed α helix, left handed
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α helix, & minor helical forms), sheet like structures (antiparallel β sheet & parallel β sheet), a triple helix
present in glycine rich proteins, and turns or bends.
THE α HELIX - The first 2° structure to be discussed is the right handed α helix. Since the amino acids in
proteins are in the L-configuration and since L-amino acids do not form stable left handed α helices, the
right handed α helix is the most prevalent and stable form of helical structure in naturally occurring proteins.
The α helix is a spiral that turns in a right handed direction. There are 3.67 (3 ⅔) amino acids per turn of the
α helix. Peptide bonds of the protein backbone are oriented so that the amido group and the carbonyl group
are parallel to the long axis of the helix. The carbonyl groups point toward the carboxyl end of the helical
region and the amido groups point toward the amino end. Hydrogen bonds between the carbonyl oxygen
and the amido hydrogen four amino acids upstream, toward the C-terminus of the helix, stabilize the helical
structure (the carbonyl oxygen of amino acid 1 is hydrogen bonded to the amido hydrogen of amino acid 5,
2 to 6, etc.). The hydrogen bonds that stabilize the helix are straight, strong, and parallel to the long axis of
the helix.
The side chains of the amino acids project from the surface of the helix, they lie perpendicular to the long
axis of the helix. Side chains play an indirect role in destabilizing or stabilizing the α helix. Proline and
glycine are helix breakers. The cyclic nature of the proline side chain puts a “kink” in the helix disrupting it
and the conformational flexibility of glycine destabilizes the helix. Large, bulky side chains next to each
other in the primary structure tend to destabilize the α helix because of steric hindrance. Adjacent side
chains carrying like charge, e.g. a string of lysine residues, disrupt the helix by electrostatic repulsion. Side
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chains of opposite charge or two aromatic side chains separated by 3 or 4 amino acids can interact with each
other and tend to add stability. The first four amido hydrogens and the last four carbonyl oxygens do not
have hydrogen bonding partners within the helix. To prevent the helix from “fraying at the ends” the
polypeptide chain usually folds to provide appropriate hydrogen bonding partners for these groups This
folding is called HELIX CAPPING. For example the hydroxyl group of a serine residue can, when the protein
is properly folded, be positioned to hydrogen bond with one or more of the first four amido hydrogens.
THE β SHEET - The β sheet is a more extended conformation. In the β sheet conformation two or more
strands of polypeptide chain run adjacent to each other. The carbonyl oxygen and the amido hydrogen of
the peptide bond lie in the plane of the β structure. Hydrogen bonds between the amido hydrogen and the
carbonyl oxygen on neighboring strands stabilize the structure. The R groups of the amino acids alternate
above and below the plane of the β sheet. This structure is sometimes called a β Pleated Sheet because the
polypeptide backbone takes on a pleated structure.
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The polypeptide strands that make up this structure can run in the same or PARALLEL direction or they can
run in the opposite or ANTIPARALLEL direction. In theory, two strands of β structure should be stable since
this arrangement allows for stable hydrogen bonding between the strands. In actuality stable two stranded β
structures are rarely found in proteins, rather it appears that at least three strands of antiparallel β sheet is
required to form a stable structure and between five and eight strands are necessary to form a stable parallel
structure. More strands are needed in the parallel structure because the H bonds are at a less than optimal
angles and distances.
THE β BULGE - The β bulge is a small piece of non-repetitive structure that occurs by itself, but most often it
occurs as an irregularity in antiparallel β structures. The β bulge is two strands of a multi-stranded
antiparallel β sheet in which one stand, the long strand, contains one more amino acid than the other strand,
the short strand. The extra amino acid is accommodated in the long strand by creating a bulge in the long
strand and slightly bending the short strand. This structure introduces a gentle bend in the polypeptide back
bone.
TURNS - In a turn or bend the direction of the polypeptide strand changes direction by 180°. In a β turn the
180° change in direction occurs over the span of 4 amino acids. The carbonyl oxygen of the first amino acid
of the turn is hydrogen bonded to the amido hydrogen of the fourth amino acid. This hydrogen bond
stabilizes the β turn.
- Type I β turn usually (>90%) contains proline at position 2. Position 3 can be any amino acid.
- Type II β turn does not contain proline, but it always contains a glycine in position 3.
Strands of antiparallel β sheet are often looped together by β turns, but a turn can join two helical segments
or join a helical region to a sheet structure.
AA4
AA3
AA2
AA1
AA4
AA4
N
|
H
AA3
O
||
C
Gly
AA2
Pro
AA1
6
AA1
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Tertiary Structure
The folding of a single polypeptide chain in three dimensional space is Tertiary Structure. Tertiary
structures involves long range interactions within the polypeptide. The protein folds upon itself resulting in
a tight compact shape, a conformation, that is at an energy minimum and an entropy maximum. This low
energy form occurs when the amino acid side chains have made a maximum number of interactions.
Tertiary structure is stabilized by four weak intermolecular interactions and, by possibly, one covalent
interaction. These interactions are:
1. Hydrophobic Interactions. These interactions occur between the side chains of the non polar amino
acids. Hydrophobic interactions tend to cluster the non polar amino acid side chains into the interior of
the molecule as far away from polar water as possible.
2. Hydrogen Bonds. In tertiary structure hydrogen bonds form between most of polar groups on the
molecule. They can form between:
a. the side chains of the uncharged polar amino acids, e.g. Ser can H bond to Asn
b. between the uncharged polar amino acid side chains and the carbonyl oxygen and/or the amido
hydrogen of peptide bonds not involved in 2° structures, e.g., the helix capping phenomenon
c. between uncharged polar amino acid side chains and acidic and/or basic amino acid side chains, e.g.,
Thr can H bond to Glu.
3. Salt Bridges. Salt bridges are electrostatic interactions between the negative charge on acidic amino
acid side chains and the positive charge on basic amino acid side chains.
4. Hydration. Structured water interacting with the polar groups on the surface of the protein. This water
“weakens” the interactions between polar groups on the protein surface increasing the “flexibility” of the
molecule.
5. Disulfide Bonds. This is the only covalent interaction that stabilizes tertiary structure. It occurs after the
protein has correctly folded and only if two cysteine side chains are appropriately positioned to form the
bond.
O
C
HC
NH
NH
H2
C
SH
+
HS
H2
C
[O]
CH
C
O
O
C
HC
NH
NH
H2
C
S
S
H2
C
CH
C
O
Structural Motifs
STRUCTURAL MOTIFS are combinations of secondary structures that have been found to occur commonly in
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proteins. They are regions of 2° structure that interact with each other locally rather than globally.
Structural Motifs are usually not structurally stable by themselves, i.e., they fall apart if removed from the
environment of the larger protein molecule.
Some common Structural Motifs include:
Helix-Loop-Helix
βαβ Unit
Hairpin
Greek Key
Note: The Helix-Loop-Helix is also called the α-α Corner
β Meander
α/β Barrel
Domains
DOMAINS are discrete independently folded units within the 3° structure of a protein. Domains are often
combinations of several structural motifs. They are independently stable and usually perform a specific
function within the protein molecule. Domains within different proteins show a low to moderate degree of
sequence homology within their 1° structures. However, they contain a very high degree of structural
homology, containing similar secondary structures and structural motifs. The similar structure makes sense
when one considers that domains in different proteins often perform similar functions. Domains appear to
have arisen via duplication of some ancestral gene. Some examples of domains include the Nucleotide
Binding Domain (or the Rossman Fold), the Sushi Domain plays a role in protein-protein binding, the
Kringle Domain often plays a role in protein-ligand and/or protein-protein binding, and the Epidermal
Growth Factor Domain may be / is involved in interactions between extracellular proteins and the cell
surface. The epidermal growth factor domain may have biological (growth factor) activity when cleaved
from the larger protein molecule.
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Quaternary (4°) Structure
A large number of proteins acquire biological function once they have attained tertiary structure. Some
proteins have biological function only when two or more polypeptide chains interact with / bind to each
other to form a multimeric protein. Individual polypeptide chains that make up this multimeric protein are
called PROTOMERS or SUBUNITS. The arrangement of subunits in space to form a functional multimeric
protein is Quaternary (4°) Structure. The protomers forming this multimeric protein can be identical, having
the same 1°, 2°, and 3° structure (homomultimeric or oligomeric proteins), or they can be different, having
different 1° structures which results in the subunits folding into different 2° and 3° structures
(heteromultimeric proteins). Hemoglobin is a heterotetramer composed of two α-subunits and two β-chains.
The forces that stabilize 4° structure, the forces that hold the subunits together, are the same forces that
stabilize 3° structures; hydrophobic interactions, hydrogen bonds, salt brides, hydration, and disulfide
bonds.
Non-Regular Non-Repeating Structures
When a protein folds into its higher order structures (2° & 3°) some of the molecule folds into regular
repeating structures; helices, sheets, and turns. The remaining regions of the molecule assumes no definite
repeating structure. These regions used to be called Random Coils or Random Structure. The current term
for these areas of a protein is NON-REGULAR NON-REPEATING STRUCTURES. In the final three dimensional
molecule these stretches of the polypeptide are ordered structures locked into a specific conformation. They
are held in this ordered conformation by the weak noncovalent forces and/or disulfide bonds that stabilize
the higher order structures. In some proteins segments at the amino terminal and/or the carboxyl terminal
end of the molecule are truly random and they exist in many alternative conformations.
Intrinsically Unstructured Proteins or Natively Unfolded Proteins
Recent studies on the human PROTEOME have resulted in the discovery of a group of low molecular weight
proteins that are fully unfolded in their “native” state. {The PROTEOME is the entire complement of proteins
that is or can be expressed by a cell, tissue, or organism.} These proteins are called INTRINSICALLY
UNSTRUCTURED PROTEINS (IUP) or NATIVELY UNFOLDED PROTEINS. Amino terminal and/or the carboxyl
terminal regions (>30 & <100 amino acids) of natively unfolded structure have also been identified in
several large folded proteins. IUP’s lack specific secondary and tertiary structure and exist in the cell as a
stable mixture of conformations. Common features of the IUP’s include:
1. low sequence complexity.
2. low proportion of hydrophobic and/or bulky amino acids - Val, Leu, Ile, Met, Phe, Tyr, & Trp; these
hydrophobic amino acids form the “central core” of globular proteins.
3. high proportion of certain polar or charged amino acids - Gln, Ser, Glu, & Lys
4. high proportion of amino acids with a high degree of conformational flexibility - Gly, Ala, & Pro
NATIVELY UNFOLDED PROTEINS take part in signaling events by binding to numerous other proteins, nucleic
acids, or membrane components. When the unfolded protein / region binds its partner it often assumes a
stable secondary and tertiary structure. The extended unfolded structure increases the surface area and
flexibility of the protein so it can more easily find its binding partner. When bound to their protein partners
the NATIVELY UNFOLDED PROTEINS may act as an inhibitor of that protein or it may act as a scaffold holding
several proteins together in some “signaling” pathway.
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The Folding Process
Looking at an individual polypeptide chain one would expect the unfolded state to be favored since in this
form the molecule has the most internal entropy, it is the most random. If this were true the folding process
would not be spontaneous, the folding process would have a +ΔG, and a constant supply of energy would be
required to fold the molecule and keep it folded. However, within the system of the cell, protein folding is a
spontaneous process, the overall process has a –ΔG. When charged and/or polar side chains form
energetically favorable interactions with each other in the folded molecule the enthalpy (H) of the system
decreases slightly (small –ΔH). But, these enthalpy decreases are not large enough to overcome the
unfavorable entropy change that the molecule undergoes when it is folded into a single conformation. What
then drives the folding process? The main driving force in the formation of 3° (and 4°) structure is the
HYDROPHOBIC EFFECT or HYDROPHOBIC INTERACTIONS. As a protein assumes its 3° (and 4°) structure it
folds so that the hydrophobic side chains cluster in the interior of the molecule where they can interact with
each other. When the hydrophobic side chains interact with each other, water is released from the organized
clathrate like structures that surround them in the extended molecule. This released water greatly increases
the entropy of the system; the water of the cell is much more random. Interactions between polar side
chains (i.e., hydrogen bonds between uncharged side chains, ion-dipole interactions between charged and
uncharged polar side chains, and/or salt bridges between charged side chains) further increases the entropy
of the system by releasing the water that was initially hydrogen bonded to these polar groups. The stability
of a tertiary and/or quaternary folded protein depends upon the interplay of three factors:
1. The unfavorable entropy change of the protein molecule as it folds. The protein would favor a
random structure instead.
2. The favorable enthalpy arising from intramolecular side chain interactions.
3. The favorable entropy change arising from burying the hydrophobic groups within the molecule and
the concomitant release of structured water.
Mechanisms of Protein Folding
Protein folding is a spontaneous process, but it is not a
random event. A protein does not “try out” all of the
possible allowed conformations before “deciding” on the
correct one. Folding appears to occur in a series of
distinct stages. Not all of the principles that guide the
folding process have been elucidated, but it appears that
the folding process is hierarchical:
1. Secondary structures form first, driven by
sequences of amino acids in the 1° structure that
favor certain secondary structures.
2. Structural motifs form as more of a localized area
assumes its 2° structure.
3. The protein collapses into a MOLTEN GLOBULE
mediated by hydrophobic interactions between
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the non polar amino acid side chains. The molten globule state has some secondary structures and
motifs, but many of the amino acids are not yet fixed in the native state
4. Domains form from collections of supersecondary structures as the molten globule continues to
rearrange and the side chains of the amino acids become fixed in their native location.
5. The polypeptide assumes its correctly folded tertiary structure.
6. If necessary, correctly folded polypeptides (subunits) interact to form multimeric proteins.
Molecular Chaperones
Not all proteins fold spontaneously into their correct native state. From 10 to 30% of cellular proteins
require the aid of a group of specialized proteins called MOLECULAR CHAPERONES. Molecular Chaperones
appear to be present in all living organisms. Quite often chaperones are required for the correct folding and
subunit association of proteins with 4° structure. For example, the α-hemoglobin chain requires ALPHA
HEMOGLOBIN STABILIZING PROTEIN (AHSP) during the assembly process. The α-hemoglobin chain is
synthesized first and in excess over the β-chain. AHSP prevents the α-chain from aggregating and
precipitating while partner β-chains are being synthesized.
40
ATP
70
P
AT
P
AT
AT
P
ATP
AT
P
AT
P
ATP is hydrolyzed to ADP and Pi
Energy released used to fold protein
ATP exchanged for ADP
Hsp40 & Hsp70 released
ATP
ADP ADP
ADP
ADP ADP
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A second class of chaperones help to fold highly hydrophobic proteins or refold partially unfolded
molecules. This class consists of proteins from the HEAT SHOCK PROTEIN (Hsp) family. Many of the heat
shock proteins act as chaperones. Two of them, Hsp 70 (70000 MW) and Hsp 40 (40000 MW), illustrate
how they function (see above). Hsp 40 binds to the unfolded polypeptide, followed by Hsp 70 and ATP.
Hsp 70 binds to hydrophobic regions of unfolded polypeptides preventing inappropriate aggregation. The
ATP is hydrolyzed to ADP and PO4–3, and the energy released by the ATP hydrolysis is used to fold the
protein. The ADP is exchanged for ATP and the folded protein is released. If folding is not complete, the
process repeats or the unfolded protein is moved to a CHAPERONIN complex.
ATP
ADP
ADP
ADP
ADP
Protein Folded
ATP
ATP
ATP Hydrolysis
ATP
GroEL and GroES
7 ADP
(Partial
Protein
ADP
ADP
ATP
GroES
Folded
eleased
ATP
ATP
ATP
ADP
ADP
ADP
ADP
7 ATP
7 ADP
ATP
ATP
ADP
ADP
ADP
ATP
ATP
ATP
ADP
ATP
ATP
ADP
ADP
ATP
7 ATP
ATP Hydrolysis
Protein Folded
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CHAPERONINS are elaborate protein complexes composed of two stacked rings composed of 6, 7, or 8 Hsp
60 subunits. Bacterial chaperonins have a third ring composed of different subunits that function as a “lid”;
in eukaryotes the “lid” is composed of a domain donated by the Hsp 60 subunits that comprise the rings.
The best characterized CHAPERONIN is the GroEL/GroES of E.coli. GroEL is 14 Hsp 60 polypeptides
associated to form 2 rings, each containing 7 Hsp 60 subunits. GroES is a heptamer “lid” structure
comprised of Hsp 10 polypeptides that closes one end of the GroEL rings. The unfolded protein enters
GroEL at its open end. ATP binds to the GroEL ring with the peptide and ADP dissociates from the “empty”
ring causing the “movement of GroES from the “empty” ring to the “full” ring. The ATP bound to GroEL is
slowly hydrolyzed to ADP and PO4–3, and the energy released causes conformational changes in the GroEL
ring containing the folding peptide. The conformational changes in GroEL are communicated to the
encapsulated protein forcing it to assume new conformations and fold properly. If folding is not complete,
the process repeats. In addition to aiding the folding process of some Molecular Chaperones escort selected
proteins to their specific cellular location. A specific enzyme associated with chaperones in both
prokaryotes and eukaryotes catalyzes the formation / rearrangement of disulfide bonds within proteins. This
enzyme is Protein-Disulfide Isomerase.
Classification of Proteins Based on Their Final Folded Form
Proteins can be divided into three major classes based upon their final folded shape, their physical
properties, and their chemical properties.
FIBROUS PROTEINS are long rod like molecules that are insoluble or only slightly water soluble. They are
physically tough and are components of structures such as skin, cartilage, tendons, bones, hair, ligaments,
hooves, and claws.
GLOBULAR PROTEINS are spherical in shape and usually very water soluble. They have a hydrophobic
interior surrounded by a hydrophilic exterior.
MEMBRANE PROTEINS are embedded in or pass through cellular membranes. Membrane proteins fold so
that there are at least two distinct domains in the molecule. There is a hydrophobic region that interacts with
the hydrophobic membrane lipids and one or more polar regions that interact with intracellular and/or
extracellular water.
Conjugated Proteins
Conjugated proteins contain nonprotein components integral to their structure and necessary for their
function. The nonprotein part is called a PROSTHETIC GROUP. The protein with out its necessary prosthetic
group is termed the APOPROTEIN. The intact functional protein, the apoprotein with its required prosthetic
group, is the HOLOPROTEIN. Conjugated proteins include:
GLYCOPROTEINS - the prosthetic groups are carbohydrates. The carbohydrate moiety is linked to the protein
by covalent bonds involving the hydroxyl group of serine or threonine. These are the O-Linked
glycoproteins. Alternatively, the carbohydrate can be linked to the protein by covalent bonds involving the
amide nitrogen of asparagine side chain. These are the N-linked glycoproteins.
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LIPOPROTEINS - the prosthetic groups are lipids.
NUCLEOPROTEINS - the prosthetic groups are nucleotides or nucleic acids.
PHOSPHOPROTEINS - have phosphoryl (phosphate) groups in ester linkage to hydroxyl groups on the protein.
METALLOPROTEINS - contain metal ions attached either by ionic interactions or by coordinate covalent
bonds.
Denaturation
A good deal of what is known about the native conformation of proteins came from DENATURATION studies.
When a protein is denatured the native conformation, the higher level structures; secondary, tertiary and
quaternary structures; are destroyed. The primary structure of the protein is unchanged. From denaturation
studies the amount and kind of secondary structures, the number and types of supersecondary structures, the
number of domains, and the stability of individual domains can be determined.
A variety of things denature proteins:
• Heat increases the kinetic energy within a protein molecule and this increased kinetic energy disrupts
the weak interactions that stabilize the higher levels of protein structure.
• Physical Agitation increases the kinetic energy within the protein and disrupts the weak interactions
within the molecule. Shear forces present during the agitation also physically disrupts the weak
intramolecular interactions.
• pH alters the ionization state of side chains and disrupts salt bridges that stabilize the protein.
• Detergents increase the solubility of hydrophobic side chains in water. This literally turns the protein
inside-out.
• Nonpolar Organic Solvents such as alcohol, ether, benzene, etc., increase the solubility of the
hydrophobic side chains. They too turn the protein inside-out.
• Chaotropic Agents such as urea or guanidinium hydrochloride disrupt hydrogen bonds within the
protein and decrease the polarity of water which increases the solubility of the hydrophobic side chains
in water.
• Heavy Metals, such as Hg, Cu, Fe, Cd, etc., denature proteins by a variety of mechanisms. The high
charge density on the metal ions disrupt salt bridges within the molecule. Fe3+ and Cu2+ can oxidize
sulfur containing groups to sulfonic acids and/or sulfones. Mercury (the pure metal) binds to the
sulfur of cysteine side chains. This can disrupt hydrogen bond formation, it can cause abnormal
disulfide bond formation, it can prevent normal disulfide bond formation in the molecule, and/or it can
break disulfide bonds that should be present for stability.
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Protein Structure - Some Examples
COLLAGEN is the most abundant protein in vertebrates. It is part of bone, teeth, ligaments, tendons, and
skin. There are, at last count, 23 different types of collagen coded for by 33 different genes. Each of the
different collagens have a slightly different structure and function. Some collagens fold into fibers others
fold into sheets. All types of collagen are rich in the amino acids Gly, Pro, and Lys. They do not contain
Cys. The primary structure of collagen contains the repeating sequences
-Gly-Pro-X- or -Hyp-Gly-Xwhere Hyp is 4-Hydroxyproline. X is often Lys and some of the lysine residues are modified to 5Hydroxylysine.
Protropocollagen
The appropriate genes are transcribed and processed in the nucleus, then transported to and translated in the
cytoplasm {transcription and translation will be examined in detail subsequently}. The polypeptide released
from the ribosome in the cytoplasm is called Protropocollagen, it is the monomer unit of the collagen
molecule. The Protropocollagen polypeptide contains an amino terminal globular domain, the extended
region containing the -Gly-Pro-X- or -Hyp-Gly-X- repeats that will become Collagen, and a carboxyl
terminal globular domain. Protropocollagen is transported into the smooth endoplasmic reticulum where the
addition of the hydroxyl group to proline and lysine occurs. The hydroxylation reactions requires ASCORBIC
ACID (Vitamin C), and is catalyzed by the enzymes Prolyl Hydroxylase or Lyslyl Hydroxylase, respectively.
A lack of Vitamin C in the diet results in the deficiency disease Scurvy. The symptoms of scurvy in adults
include spongy gums, loosening of the teeth, and bleeding into the skin and mucous membranes. Bone
development in children is abnormal resulting in bowed legs and/or stunted growth.While the post
translational modifications are occurring the carboxyl terminal domains associate with each other. After a
sufficient number of proline residues have been hydroxylated (at least 90 proline residues and 10 lysine
residues per chain) the triple helical structure will form starting at the associated carboxyl termini and
extending in a zipper like fashion to the amino terminal globular domain. Heat Shock Protein 47 (Hsp 47) is
a specific molecular chaperone in the ER that stabilizes the forming helical structure.
N- Terminal
Domain
Collagen
C- Terminal
Domain
Tropocollagen
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The triple helical form is termed Tropocollagen and they are transported from the ER into the Golgi and
secreted into the extracellular space. In the extracellular space a specific Troprocollagen aminopeptidase
and a specific Troprocollagen carboxypeptidase cleave the globular domains giving rise to the mature
collagen molecule. Removal of the globular domains decreases the solubility of the collagen molecule
dramatically. In the extracellular space, the molecules of mature collagen assemble spontaneously into
quarter-staggered fibrils. This assembly is directed by the presence of clusters of hydrophobic and charged
amino acids on the surface of the molecules. During fibril formation, some lysyl residues are deaminated by
lysine oxidase, which removes the epsilon amino group from the side chain, forming an aldehyde group.
These aldehydes associate spontaneously with epsilon amino group side chains on other molecules, forming
interchain cross links. These cross links increase the strength of the fibrils.
MYOGLOBIN and HEMOGLOBIN - Typical Globular Proteins
Myoglobin was the first globular protein to have its complete three dimensional structure solved by X-ray
crystallography. Myoglobin is a storage protein. It reversibly binds and stores O2 in muscle tissue. The
protein contains 153 amino acids and folds into eight short α-helical segments joined by short loops of
peptide (helix-loop-helix supersecondary structure). In a hydrophobic pocket between the E and F helix a
molecule of Heme is bound. Heme is Protoporphorin IX with a ferrous (Fe++) ion bound to it by 5
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coordinate covalent bonds. Oxygen reversibly binds to the Fe++ ion by a sixth coordinate covalent bond.
There are numerous contacts between the side chains of the amino acids within the helical segments. These
contacts hold the molecule in its native conformation.
Hemoglobin is a transport protein. It carries O2 from the oxygen rich lungs to the O2 poor tissues.
Hemoglobin is a multimeric protein, it has 4° structure. It is composed of two α and two β subunits. The 1°
structure of the α subunits is different from the 1° structure of the β subunits. However, the 1° structure of
the α and β subunits, as well as their 2° and 3° structures, are similar to that of myoglobin; they show a high
degree of structural homology - proteins with similar functions often have similar structures. The four
subunits are directed at the corners of a tetrahedron. The α1β 1 subunits act as a pair as do the α2β 2 subunits.
Each of the subunits of hemoglobin contain a molecule of heme between the E and F helix and each binds
one oxygen molecule reversibly at the heme, for a total of 4 oxygen molecules per hemoglobin molecule.
Transmissible Spongiform Encephalopathies - Prion Related Diseases
One of the Diseases of Incorrect (Abnormal) Protein Folding
Creutzfeldt-Jakob disease, Kuru, Gerstmann-Sträussler syndrome, and Fatal Familial Insomnia in humans as
well as Scrapie in sheep and goats, Bovine Spongiform Encephalopathy (Mad Cow Disease), and Chronic
Wasting Disease in deer and elk, are fatal neurodegenerative diseases that are part of the group of
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSEs), or PRION RELATED DISEASES. Vacuolation of
neurons, astroglyosis, and the accumulation of the abnormal, protease-resistant prion protein in the central
nervous system characterize TSE. Several unprecedented scientific findings on these diseases directly
confront some of the most popular dogmas of biology. These heretical findings include:
1. disease transmission by an ‘infectious protein’ in the absence of nucleic acid (DNA or RNA);
2. the folding of a protein in several different conformations with distinct biological properties;
3. the transmission of biological information by the replication of protein conformation.
These findings have given support for an entirely novel disease mechanism, involving transmission of
PROTEIN MISFOLDING.
The human prion protein (PrP) is 253 amino acids long. It is glycosylated near the amino terminus and it is
embedded into the extracellular surface of the cell membrane by a phosphatidylinositol-glycosyl link (a lipid
linked protein). Normal prion protein is expressed in the brain and other tissues of healthy animals and
humans. The function of PrP is for the most part unknown. However, there are some data that suggests it
plays a role in cell maturation To differentiate the normal protein from the one present in infectious
material, they are termed PrPC (Cellular) and PrPSc (Scrapie associated), respectively.
The two proteins are identical in size, but PrPSc is insoluble, not easily denatured, and resistant to protease
digestion. PrPSc has a high tendency to aggregate forming Amyloid (a generic term used to describe fibrillar
protein aggregates that stain pink with common histological stains). Techniques that allow for the
determination of secondary structure have shown that the α helical content of PrPC is 40%, with little or no β
sheet, whereas PrPSc contains 50% β sheet and only 20% α helix (see above).
The initial PrPSc ‘infectious’ molecules appear to arise from an amino acid change in the primary structure
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of PrPC. These amino acid substitutions (mutations) can be familial or they can arise spontaneously. The
following amino acid substitutions have been reported: Pro102→Leu, Pro105→Leu, Ala117→Val,
Gly131→Val, Asp178→Asn, Val180→Ile, Thr183→Ala, Thr188→Lys, Thr188→Arg, His187→Arg,
Phe198→Ser, Glu200→Lys, Arg208→His, Val210→Ile, Gln217→Arg, and Met232→Arg. The region of
the protein from Amino Acid 23 to 144 appears to contain the amyloidogenic properties of the abnormal
molecule.
Numerous findings suggest that PrPSc can replicate itself at the expense of normal PrPC by transferring its
toxic conformation to the normal protein. Prion replication is hypothesized to occur when PrPSc interacts
specifically with normal PrPC, catalyzing its structural conversion to the pathogenic form of the protein.
PrPSc binds to PrPC and acts as a template to cause the refolding into the toxic abnormal conformation. An
additional protein factor termed Protein X has been postulated to be involved in the PrPSc mediated
refolding of PrPC. As PrPSc accumulates in the brain (because it cannot be broken down by cellular
proteases?) the symptoms of the disease begin to manifest themselves.
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