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

1 ©Kevin R. Siebenlist, 2015

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


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.

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


C NH

C

C

O

CarbonylOxygen

AmidoHydrogen

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


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.


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.

AA1

AA2

AA3

AA4

O||C

N|H

AA1

Pro

AA3

AA4

AA1

AA2

Gly

AA4


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.

Structural Motifs

STRUCTURAL MOTIFS are combinations of secondary structures that have been found to occur commonly in

C

HCH2C SH

NH

O

HSH2C CH

NH

C O

C

HCH2C S

NH

O

SH2C CH

NH

C O

+[O]


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:

Note: The Helix-Loop-Helix is also called the α-α Corner

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.

Helix-Loop-Helix βαβ Unit Hairpin Greek Key

β Meander α/β Barrel


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.


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


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.

ATP

40

70

ADP

ADP ADP

ADP

ADP

ATPATP

ATP

ATP

ATP

ATP

ATP is hydrolyzed to ADP and Pi

Energy released used to fold protein

ATP exchanged for ADP

Hsp40 & Hsp70 releasedATP


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.

GroEL and GroES

7 ATP

7 ADP

GroES

7 ATP

(PartialProtein

AD

P

AD

P

AD

P AD

P

AD

P

AD

P

AD

P AD

P

ATP

ATP

ATP AT

P

AD

P

AD

P

AD

P AD

P

AD

P

AD

P

AD

P AD

P

ATP

ATP

ATP AT

P

ATP HydrolysisProtein Folded

7 ADP

ATP

ATP

ATP AT

P

Foldedeleased

ATP

ATP

ATP AT

PATP HydrolysisProtein Folded


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.


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.


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-X-

where Hyp is 4-Hydroxyproline. X is often Lys and some of the lysine residues are modified to 5-Hydroxylysine.

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.

Protropocollagen

Tropocollagen

Collagen

N- TerminalDomain

C- TerminalDomain


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


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


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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