Chapter 5 The Three-dimensional Structure of Proteins

1. Early studies on the peptide (protein) structure

1.1 The peptide (O=C-N-H) bond was found to be shorter than the C-N bond in a simple amine and atoms attached are coplanar.

1.1.1 This was revealed by X-ray diffraction studies of amino acids and of simple dipeptides and tripeptides.

1.1.2 The peptide (amide) bond was found to be about 1.32 Å (C-N single bond, 1.49; C=N double bond, 1.27), thus having partial double bond feature (should be rigid and unable to rotate freely).

1.1.3 The partial double bond feature is a result of partial sharing (resonance) of electrons between the carbonyl oxygen and amide nitrogen.

1.1.4 The atoms attached to the peptide bond are coplanar with the oxygen and hydrogen atom in trans positions.

1.2 X-ray studies of -keratin (the fibrous protein making up hair and wool) revealed a repeating unit of 5.4 Å (Astury in the 1930s).

2. The likely regular conformations of protein molecules were proposed before they were actually observed!

2.1 This was accomplished by building precise molecular models.

2.1.1 Experimental data (from X-ray studies) were closely adhered, interpreted.

2.1.2 Single bonds other than the peptide bond in the backbone chain are free to rotate.

2.2 The simplest arrangement of the polypeptide chain was proposed to be a helical structure called -helix (Pauling and Corey, 1951)

2.2.1 The polypeptide backbone is tightly wound around the long axis (rodlike).

2.2.2 R groups protrude outward from the helical backbone.

2.2.3 A single turn of the helix (corresponding to the repeating unit in a-keratin) extends about 5.6 Angstroms, including 3.6 residues (each residue arises 1.5 Å and rotate 100 degrees about the helix axis).

2.2.4 The model made optimal use of internal hydrogen bonding for structure stabilization.

2.2.5 Each carbonyl oxygen of the residue n is hydrogen bonded to the NH group of residue (n+4).

2.2.6 The residues forming one -helix must all be one type of stereoisomers (either L- or D-).

2.2.7 L amino acids can be used to build either right- or left-handed -helices (the helix spiraling away clockwise or counterclockwise respectively).

2.3 -pleated sheet was proposed to be the more extended conformation of the polypeptide chain.

2.3.1 The conformation is formed when two or more almost fully extended polypeptide chains are brought together side by side.

2.3.2 Regular hydrogen bonds are formed between the carbonyl oxygen and amide hydrogen between adjacent chains (look like a zipper).

2.3.3 The axial distance between the adjacent amino acid residues is ~3.5 Angstroms.

2.3.4 The planes of the peptide bonds arrange as pleated sheets.

2.3.5 The R groups of adjacent residues protrude in opposite directions.

2.3.6 The adjacent polypeptide chains can be either parallel (the same direction) or antiparallel (the opposite direction).

3. Protein architecture can be understood at different levels.

3.1 Each protein usually has one native conformation

3.1.1 Under physiological conditions of solvent and temperature, each protein folds spontaneously into one three-dimensional conformation, called the native conformation.

3.1.2 This conformation is usually thermodynamically the most stable (having the lowest Gibb’s free energy), and predominates among the innumerable theoretically possible ones.

3.1.3 Usually only the native conformation is functional.

3.2 Protein structures have conventionally been considered at four different levels.

3.2.1 The primary structure is the amino acid sequence (including the locations of disulfide bonds).

3.2.2 The secondary structure refers to the regular, recurring arrangements of adjacent residues resulting mainly from hydrogen bonding between backbone groups, with a-helices and b-pleated sheets being the two most common ones.

3.2.3 The tertiary structure refers to the spatial relationship among all amino acid residues in a polypeptide chain, that is, the complete three-dimensional structure.

3.2.4 The quaternary structure refers to the spatial arrangements of each subunit in a multisubunit protein, including nature of their contact.

3.3 Studies of protein conformation, function, and evolution have revealed importance of two other levels of organization.

3.3.1 The supersecondary structure refers to clusters of secondary structures that repeatedly appear in different proteins.

3.3.2 The already identified supersecondary structures include mainly motif, Greek key motif, -hairpin loop, four-helix-bundle, …etc.

3.3.3 Supersecondary structure motifs are usually also folding motifs of proteins. (a conjecture, Not completely established experimentally).

3.3.4 A compact region (usually include less than 200~400 residues) that is a distinct structural unit within a larger polypeptide chain is called a domain.

3.3.5 Many domains fold independently into thermodynamically stable structures, and sometimes, have separate functions.

Structural domains in the polypeptide troponin C, two separate calcium-binding domains

Repeated usage of a pattern

deoxyhemoglobin

Quanternary structure: macromolecular assembly

4. The theoretically allowed conformations of peptide main chains can be predicted by the Ramachandran plot

4.1 The backbone conformation of a peptide bond can be defined by two sets of rotation angles.

4.1.1 The rotation angles around the N-C bonds are labeled as phi, and around C-C bonds are psi.

4.1.2 By convention, both phi and psi are defined as 0 degree in the conformation when the two peptide planes connected to the same a carbon are in the same plane.

4.1.3 In principle, phi and psi can have any value between -180 and +180 degrees.

4.1.4 The conformation of the main chain is completely defined when phi and psi are specified for each residue in the chain.

4.2 The allowed combination values for phi and psi can be graphically shown by plotting one against the other.

4.2.1 The Ramachandran plot is based on the principle that no two atoms can come together closer than the sum of their van der Waals radii (hard sphere model).

4.2.2 Many combinations of rotation angles are not allowed in peptide backbone conformation due to steric hindrance.

4.2.3 Gly residues can take up many conformations that are sterically forbidden for other residues.

4.2.4 Every possible secondary structure is described completely by the two bond rotation angles that are the same for each consecutive residue. For right-handed -helix, phi=-60 and psi=-45 to -50; for -pleated sheets, phi is around -120 and psi 120.

Phi=0, psi=0

5. -helices and -pleated sheets were confirmed to be common secondary structures in proteins

5.1 Both -helix and -pleated sheet were later found to be existing in proteins.

5.1.1 The existence of -helices in protein was confirmed (6 years after its prediction) by the determination of the X-ray structure of myoglobin.

5.1.2 Right-handed -helices widely exist in proteins.

5.1.3 -pleated sheets are also frequently found in globular proteins (-keratin, immunoglobular domain, (-barrel).)

5.2 The amino acid sequence affects the stability of -helical structures.

5.2.1 Additional interactions (e.g., electrostatic interactions, hydrophobic interactions, steric hindrance) between amino acid side chains can stabilize or destabilize the regular secondary structure.

5.2.2 Critical interactions occur between side chains several residues (2-4) away on a helix structure.

5.2.3 Pro is rarely found in -helices due to its lack of rotation around the N-C bond and inability to form hydrogen bond from its amide nitrogen.

5.2.4 Negatively charged residues at the N-terminal end of a helical segment will have a stabilizing effect on the positive charge of the helix electric dipole. (Asp, Glu)

5.2.5 Positively charged residues will have a similar stabilizing effect on the C-terminal end of the helical segment (negatively charged). (Arg, Lys)

5.3 turn (hairpin turn) is also a common secondary structure found where a polypeptide chain abruptly reverses its direction.

5.3.1 It often connects the ends of two adjacent segments of an antiparallel -pleated sheet.

5.3.2 It is a tight turn of ~180 degrees involving four amino acid residues.

5.3.3 The essence of the structure is the hydrogen bonding between the C=O group of residue n and the NH group of the residue n+3.

5.3.4 Gly and Pro are often found in turns. Gly is there because it is small and flexible; Pro because the peptide bond involving Pro can assume the cis configuration, which in turn generates a tight turn on the polypeptide chain.

5.3.5 turns are often found near the surface of a protein.

5.4 Some amino acid residues are accommodated in the different types of secondary structure better than others.

5.4.1 The probability is calculated from known protein structures. It is used in predicting secondary structures.

5.4.2 Some bias or propensities can be explained easily.

5.4.3 Others are not yet understood.

With different phi, psi angles.

The crystal structure of pyruvate kinase

The two residuesthat are 2-4 residuesaway are closein space.

Helical dipolemoment

Five different kinds of contraints affectting the stability of the helix

1) The electrostatic repulsion (or attraction) between the neighboring amino acid residues with the charged R groups.

2) The bulkiness of adjacent R groups.

3) The interactions between R groups spaced 2 to 4 residues apart.

4) The occurrence of Pro and Gly residues.

5) The interaction between amino acid residues at the ends of a helix segment with the electric dipole inherent to the helix.

6. The left-handed polypeptide chains wrap together to form a right-handed triple helix in collagen protein

6.1 The amino acid sequence of collagen is revealed to be remarkable regular.

6.1.1 Nearly every third residue is Gly (X-X-G).

6.1.2 It is abundant in Pro and Hyp (hydroxylproline).

6.1.3 The sequence Gly-Pro-Hyp recurs frequently.

6.2 The helical motif of its three chains is entirely different from that of the a-helix.

6.2.1 Intrachain hydrogen bonds are absent.

6.2.2 Each of the three helices is stabilized by steric repulsion of the pyrrolidone rings of the Pro and Hyp residues.

6.2.3 The rise per residue is 2.9 Å and there are nearly 3 residues per turn.

6.2.4 Interchain hydrogen bonds are formed (main chains ?).

6.2.5 Hyp also participate interchain hydrogen bonding.

6.2.6 Collagens also contain hydroxylysine that is believed to participate in hydrogen bonding.

6.2.7 The collagen polypeptide chains within or between the triple helices are covalently cross linked through Lys or Hylys side chains.

6.2.8 Only the small Gly can fit into the crowded interior of the triple helix.

6.2.9 The superhelix provides great tensile strength with no capacity to stretch.

6.2.10 Collagen fibers have similar tensile strength as a steel wire of equal cross section.

6.3 Collagen is the most abundant protein in mammals.

6.3.1 About 25% of the total protein mass in mammals is collagen.

6.3.2 It is a major component of tendons, the extracellular matrix of the connective tissues (skin, bone matrix), and the cornea of the eye.

6.3.3 Collagen triple helices (also called tropocollagen) self-assemble in the extracellular space to form much larger collagen fibrils that further aggregate into collagen fibers.

6.3.4 The collagen triple helices are regularly staggered in fibril to give rise to the striated appearance in negatively stained electron micrograph.

6.3.5 The fibril formation involves many enzymatic steps. Deficiency of these steps generate many genetic diseases (e.g., osteogenesis imperfecta, Ehlers-Danlos syndrome, both resulted from single amino acid replacements of a Gly).

7. -keratins contain -coiled coils

7.1 -keratins are rich in hydrophobic residues.

7.1.1 Phe, Ile, Val, Met, and Ala residues are rich.

7.1.2 This makes the protein insoluble in water.

7.2 Three helical strands wrap together to form a superhelix (protofibril) in -keratin.

7.2.1 Each strand is a -helix.

7.2.2 The superhelical twisting is left-handed in -keratins (opposite to the individual strand).

7.2.3 The individual -helices are cross linked by interchain disulfide bonds.

7.3 -keratins are the main components of skin and many skin derivatives in vertebrate animals.

7.3.1 Including, e.g., hair, wool, feathers, nails, claws, quills, scales, horns, hooves, tortoise shell, and much of the outer layer of skin.

7.3.2 Usually harder a-keratins contain higher number of Cys (18% of the residues are Cys in tortoise shells and rhinoceros horns).

7.3.3 -keratins can be stretched (to twice as its original length) due to its structure springiness.

7.3.4 Permanent waving of hair is biochemical engineering, where disulfide bonds between individual chains are reduced (while the hair being heated), curled, and reoxidized (cooled at the same time).

8. Elastin molecules contain random coils and covalently cross-linked Lys residues.

8.1 The monomer elastin (called tropoelastin) is rich in Gly, Ala, and Lys residues.

8.1.1 It contains alternating Gly-rich coiled domains and Lys-rich domains.

8.1.2 Lys side chains are covalently linked by enzymes.

8.2 Elastin is important component of elastic connective tissues (lung, large blood vessels).

The combination of random coils and covalent cross linking provide the elasticity of the tissues.

9. Sperm whale myoglobin, the oxygen carrier in muscle, was the first protein to be seen in atomic detail by X-ray analysis (John Kendrew, 1950s)

9.1 The existence of a-helices were for the first time directly observed in a protein.

9.1.1 The myoglobin molecule contains eight a-helices.

9.1.2 All the a-helices are right-handed.

9.1.3 All the peptide bonds are in the planar trans configuration.

9.1.4 There is no b-pleated sheets observed in the molecule.

9.2 The myoglobin molecule has a dense hydrophobic core.

9.2.1 Many hydrophobic R groups (e.g., Leu, Val, Met, Phe) were found to be in the interior of the myoglobin molecule.

9.2.2 Hydrophobic interaction is important for the stability of the protein structure.

9.2.3 Only two hydrophilic histidine residues were found in the interior of the protein.

9.2.4 The compactness of the molecule is similar to a solid.

9.2.5 Short-range van der Waals interactions make a significant contribution to the stabilizing hydrophobic interactions due to the solid-like compactness of the molecule (residues of subtle difference were used to fill the interior of a protein neatly and thus maximize van der Waals interactions.

9.3 All but two of the polar R groups are located on the outer surface and hydrated.

9.3.1 Nonpolar residues also present on the outer surface!

9.4 Bending are made of residues or sequences that are incompatible with a-helical structure.

9.4.1 All Pro residues are found at bends.

9.5 The flat heme group (the prosthetic group) was revealed to rest in a crevice (pocket).

9.5.1 The heme group consists of a complex organic ring structure, protoporphyrin, to which is bound an iron atom in its ferrous (Fe2+) state.

9.5.2 The iron atom has six coordination bonds, with four in the plane of and bonded to the flat porphyrin molecule and two perpendicular to it.

9.5.3 One of the perpendicular coordination bonds is bound to a nitrogen atom of an interior His residue.

9.5.4 The sixth coordination serves as the binding site for O2.

9.5.5 The accessibility of the heme group to solvent is highly restricted, thus preventing the oxidation of the Fe2+ to the ferric ion (Fe3+), which is unable to bind O2.

Sir John Kendrew and Max Perutz won the Nobel Prize in Chemistry in 1962 for determining the complete atomic structure of myoglobin and hemeglobin.

10. Atomic structures of many proteins have since been determined.

10.1 Proteins differ in three dimensional structures.

10.1.1 Myoglobin represents only one of many ways that a polypeptide chain can be folded.

10.1.2 Another small heme-containing protein, cytochrome c, was found to contain only about 40% -helices, but many irregularly coiled and extended segments.

10.1.3 The heme group in cytochrome c is covalently attached to a Met sulfur and a His nitrogen atom (different from myoglobin).

10.1.4 Lysozyme, an enzyme that catalyzes the hydrolytic cleavage of polysaccharides in some bacterial cell walls, contains about 40% -helices and about 12% -sheets.

10.1.5 Ribonuclease A contains more -sheet structures.

10.1.6 Disulfide bonds were observed in lysozyme and ribonuclease A.10.2 All the water soluble globular proteins have a hydrophobic core and a mainly hydrophilic outer surface.

10.2.1 Proteins are stabilized mainly by noncovalent interactions (sometimes by disulfide bonds).

10.2.2 Each protein has a unique structure to perform its unique function.

11. Tertiary structure is determined by amino acid sequence

11.1 Loss of tertiary structure (native conformation) is accompanied by loss of function.

11.1.1 Proteins are relatively easy to lose their tertiary structures due to their marginal stability maintained by noncovalent interactions.

11.1.2 The process of total loss or randomization of three-dimensional structure of proteins is called denaturation.

11.1.3 Protein denaturation results from a change in the solvent environment that is sufficiently large to upset the forces that keep the protein structure intact.

11.1.4 Many means can cause protein to denature:Heating: unbalance the compensating entha

lpic and entropic contributions, thermal motion causes melting.

Extreme pH: upset the balance of a protein’s charge interactions.

Miscible organic solvents (alcohol and acetone): solvent polarity and hydrogen bonding.

Solutes (urea, guanidine): provide alternative hydrogen bonding.

Detergents (SDS): introducing their hydrophobic tails into the protein’s interior.

11.1.5 The mechanism of many of these denaturing processes are fully understood.

11.2 Denaturation of some proteins is reversible.

11.2.1 Some denatured globular proteins will regain their native structure and their biological activity once returned to conditions in which the native conformation is stable. This process is called renaturation.

11.2.2 The denaturation and renaturation phenomena was originally observed on ribonuclease A by chance by Christian Anfinsen (1950s).

11.2.3 Ribonuclease A became reduced and randomly coiled (denatured) in 8 M urea plus b-mercaptoethanol, with a loss of the enzymatic activity.

11.2.4 When urea and -mercaptoethanol were removed, the enzymatic activity was slowly regained (enzymatic activity was fully regained under stable conditions, with existence of trace amount of -mercaptoethanol.

11.2.5 All the physical and chemical properties of the refolded enzyme were virtually identical with those of the native enzyme.

11.2.6 Conclusion: the information needed to specify the complex tertiary structure of ribonuclease A is all contained in its amino acid sequence.

11.2.7 Subsequent studies have established the generality of this central principle of molecular biology: sequence specifies conformation.Nobel Prize in Chemistry in 1972 for Anfinsen.

11.3 The tertiary structures of proteins are not rigid.

11.3.1 Many studies have found that globular proteins have certain amount of flexibility in their backbones and undergo short-range internal fluctuations.

11.3.2 Many proteins undergo small conformational changes in the course of their biological function (e.g., O2-bound hemoglobin differs from O2-free hemoglobin, substrate binding to enzymes often cause conformational changes).

12. The polypeptide chain of a protein folds rapidly in vivo

12.1 The protein folding problem is one of the most challenging and important areas of inquiry in biochemistry.

12.1 How does the amino acid sequence of a protein specify its three-dimensional structure?

12.2 How does an unfolded polypeptide chain acquire the form of its native conformation?

12.2 Are all possible conformations searched to find the energetically most favorable one?

12.2.1 The Levinthal’s paradox: the huge difference between the calculated (theoretical) time it may take for a polypeptide to fold by random searching and the actual time it takes.

12.2.2 The cumulative selection, that is, partially correct intermediates are (recognized by nature and) retained due to sub-stability, makes the searching process much more efficient.

12.3 Protein folding is an intriguing problem for both theoreticians and experimentalists.

12.3.1 Proteins are only marginally stable. The free energy difference between the folded and unfolded states of a typical 100-residue protein is only 10 kcal/mol, meaning that correct intermediates can be easily lost.

12.3.2 The criterion of correctness is the total free energy of the transient species, not a residue-by-residue scrutiny of conformation (?).

12.3.3 Some intermediates, called kinetic traps, have a favorable free energy but are not on the path to the final folded form.

12.4 Molten globules are formed early in folding.

12.4.1 Molten gobule state contains native secondary but not tertiary structure (an experimental observation).

12.4.2 Hydrophobic collapse and acquisition of stable secondary structure are mutually reinforcing events in the formation of molten globules (synergistic, helping each other).

12.5 Partially folded intermediates can be detected, trapped, and characterized.

12.5.1 Rapid-kinetics studies, where protein secondary structures are monitored by spectroscopic methods (e.g., fluorescence, circular dichroism), can reveal the progression of distinctive intermediates during refolding processes.

12.5.2 Disulfide-bonded intermediates can be trapped covalently by blocking uncombined cysteines with iodoacetate.

12.5.3 Pulsed hydrogen-deuterium exchange can be used to monitor the acquisition of secondary structures in protein folding.

12.5.4 Our understanding of protein folding can be stringently tested by designing novel proteins with distinctive functions. For example, encouraging starts have been made in synthesizing new scaffolds, metal-binding proteins, channels, and catalysts.

12.6 Protein folding in vivo is sometimes catalyzed by isomerases and chaperone proteins.

12.6.1 The formation of correct disulfide pairing in nascent proteins is catalyzed by protein disulfide isomerase (PDI), which is especially important I accelerating disulfide interchange in kinetically trapped folding intermediates.

12.6.2 Peptidyl prolyl isomerases (PPIases) accelerate cis-trans isomerization of Pro residues during protein folding.

12.6.3 Molecular chaperones in cells facilitate the correct assembly (including folding, refolding, formation of oligomeric complexes, … etc) of other polypeptides but are not themselves part of the assembled, functional structure. Are they enzymes? Facilitated search (e.g., avoiding aggregates)? How? Certainly, they do not specify the final structure? But do they specify the path? May specify certain properties of the paths and/or intermediates. May provide an appropriate environment for folding. Many questions are yet to be answered.