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Biochemistry Chapter 6. The Three-Dimensional Structure of Proteins Mak Oi Tong. Introduction . This chapter is devoted to an examination of: the several levels of protein structure – their geometry how they are stabilized and their importance in protein function. Structure of proteins.

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biochemistry chapter 6

BiochemistryChapter 6

The Three-Dimensional

Structure of Proteins

Mak Oi Tong

introduction
Introduction

This chapter is devoted to an examination of:

  • the several levels of protein structure – their geometry
  • how they are stabilized and
  • their importance in protein function.
structure of proteins
Structure of proteins
  • Primary structure : the amino acid sequence of protein molecules.
  • Secondary structure : the local regular folding (direction) of protein molecules.
  • Tertiary structure : the final 3-dimensional (folded) form of protein molecules.
  • Quaternary structure : the arrangement of several polypeptide chains (subunits).
  • Conformation and configuration.
secondary structure regular ways to fold the polypeptide chain
Secondary StructureRegular Ways to Fold the Polypeptide Chain

Discovery of Regular Polypeptide Structures

(Figure 5.12b, Figure 6.2, Figure 6.3, Figure 6.4)

  • Bond lengths and bond angles should be distorted (changed) as little as possible shown in Fig. 5.12b.
  • No two atoms should approach one another more closely than is allowed by their van der Waals radius.
  • The amide group must remain planer and in the trans configuration.
  • Hydrogen bonds are most probably used to stabilize the peptide folding between amide protons and carbonyl oxygen.
molecular helices and pleated sheets
Molecular Helices and Pleated Sheets
  • (Figure 6.5, Figure 6.6, Table 6.1)
helix
α-Helix
  • Only right-handed helix found.
  • Hydrogen bonds are found between the amide hydrogen and carbonyl oxygen.
  • A loop of 13 atoms is formed between the hydrogen bond.
  • 3.6 amino acids per turn of helix.
  • The repeat (c) is 18 amino acid residues.
  • The (p), distance per turn, is 0.54 nm, and the rise, distance between each atom is 0.15 (0.54/3.6).
  • α-Helix is also called 3.613 helix, compared to

π-helix 4.416 and 310 helix.

  • Proline is the α-breaker.
sheet pleated sheet
β-sheet (pleated sheet)
  • Two types of β-sheet, parallel (same N to C direction) and anti-parallel (opposite N to C direction) between polypeptide chains.
  • β-Sheet contains 2 amino acid residues per turn, and cannot form hydrogen bond between amide hydrogen and carboxyl oxygen.
  • Hydrogen bonds can only formed between adjacent polypeptide chains.
  • Anti-parallel form is more stable than parallel form.
ramachandran plots figure 6 2 figure 6 8 figure 6 9 figure 6 10
Ramachandran Plots(Figure 6.2, Figure 6.8, Figure 6.9, Figure 6.10)
  • A map (Fig. 6.8) describes the backbone conformation of any particular residue in a protein corresponding to the angle Φ (phi) and Ψ (psi)
  • Rotation of the corresponding angle Φ and Ψ are given by the arrow in clockwise for +180° when looking in either direction from the α-carbon.
  • This is called Ramachandran plot after the biochemist who first found it.
  • If all AA residues in a protein have the same secondary structure (e.g. α-helix), the points for all residues would superimpose (come together at one single point).
slide19
Because of the steric effect and van der Waals radii, only a relatively small fraction of conformations is possible, the uncolored areas of the plot in Fig. 6.8.
  • Only right-hand helix is favoured because all AAs are L-form.
  • Some points of proteins will fall in the non-allowed regions because of the glycine residue.
  • The conformation Φ = 0, Ψ = 0 is not allowed in any polypeptide chain because of the steric clash between the carbonyl oxygen and amino proton.
fibrous proteins
Fibrous Proteins

Structural Materials of Cells and Tissues (Table 6.2)

  • More than 1/3 or more of the body protein in large vertebrates.
  • Functions include:
    • External protection such as skin, hair, feather and nail.
    • Structure and support, shape and form, tendons, cartilage and bone.
  • Native conformations of fibrous proteins are stable proteins after isolated and will not be denatured or unfolded.
keratins
Keratins
  • Two classes of keratin, α- and β-keratins.
  • α- Keratins is the major fibrous proteins of hair and wool.
  • Each α-keratin molecule contains over 300 residues in length and all are α-helical.
  • Two α-keratin helices bond together by their hydrophobic R-side groups.
  • The higher amounts of AAs are serine, glutamine, glutamic acid and cysteine.
  • Disulfide cross-links are found in nail and hair (fewer), and human hair for permanent wave.
keratin
β-Keratin
  • Contains β-sheet structure.
  • Are found mostly in birds and reptiles for feather and scales.
fibroin
Fibroin
  • They areall anti-parallel pleated sheet structure.
  • Comprise almostglycine, alanineand serine AA.

Gly-ala-gly-ala-gly-serine-gly-ala-ala- gly-

This alternation allows the β-sheet to fit together.

  • The structure is strong and inextensible.
  • No intrachain hydrogen bond, only interchain hydrogen bonds.
  • No cystine cross-linkage presence.
  • The fibers are very flexible, e.g. silk.
collagen figure 6 13
Collagen (Figure 6.13)
  • Collagen is about one third of total protein in large animals, and is the major constituent of tendons and skin.
  • Basic unit is tropocollagen which consists of three polypeptide chains in left-handed helix tightly coiled into a three-strand rope in right-handed helix, arranged in head to tail.
  • Tropocollagen have total about 3000 AA residues (each polypeptide has 1000 AA residues), having of molecule weight of 300,000.
  • Every third residue can be only glycine because of the bulky effect.
slide33
Proline or 4-hydroxyproline is found in tropocollagen molecule, and a repetitive sequence is present of the form gly-X-Y, where X is often proline and Y is proline or hydroxyproline.
  • Hydrogen bonds are present among the polypeptides of tropocollagen.
  • Vitamin C is required for the enzyme to catalyze the hydroxylation of proline to hydroxyproline.
  • Scurvy, a disease which is the weakening of collagen fibers caused by the failure to hydroxylate proline, is a symptom of extreme vitamin C deficiency.
slide34
Collagen cannot be stretched.
  • The toughness of collagen is due to the cross-linking of tropocollagen molecules to one another by a reaction involving lysine side chains.
  • This process continues through life, and the accumulation cross-links make the collagen steadily less elastic and more brittle, the signs of aging.
collagen synthesis figure 6 14
Collagen Synthesis (Figure 6.14)
  • Posttraslational modification is present in the collagen synthesis.
  • Procollagen is yield having about 1500 residues of which 500 are in N- and C-terminals regions that do not have the typical collagen fiber sequence.
  • The procollagen triplexes are exported into the extracellular space and the N- and C-terminals regions are cleaved off by specific proteases.
  • See Fig. 6.14 for further discussion.
e lastin
Elastin
  • Basic component of connective tissue of blood arteries and ligaments.
  • Structure is similar to collagen.
  • The basic unit of elastin fibrils is tropoelastin.
  • It contains rich in glycine and alanine, and also high % of lysine but fewer of proline.
  • The ability for stretch in elastin is caused by the special structure of desmosine cross-linked by four lysine residues to become a form of highly interconnected, rubbery network.
different folding for different functions figure 6 1 6 15 and 6 16
Different Folding for Different Functions (Figure 6.1, 6.15 and 6.16)
  • Globular proteins which are named because their polypeptide chains are folded into compact structures.
  • They are the important structures for all kinds of enzymes, hormones and other functional proteins.
  • Their tightly folded conformations are referred as the tertiary structure and are the crucial factor for the biological functions.
  • Many globular proteins carry prosthetic groups, the non-amino acid small molecules that may be noncovalently or covalently bonded to the proteins to fulfill special functions.
  • Every globular protein has a unique tertiary structure that is made up of secondary structure elements such as helics, β -sheet.
varieties of globular protein structure patterns of folding figure 6 16 6 17 6 18 and 6 19
Varieties of Globular Protein Structure: Patterns of Folding Figure 6.16, 6.17, 6.18, and 6.19)
  • Although there will be infinite number of globular protein folding, some principles of protein folding are found.
  • Many proteins are made up of a number of domains, a compact locally folded region of tertiary structure, which perform different functions of proteins.
  • Two major kinds of folding patterns, α-helic and β-sheet.
general rules of globular proteins
General rules of globular proteins
  • All globular proteins have a defined inside and outside structure, hydrophobic AAs inside and hydrophilic AAs outside.
  • β-Sheets are usually twisted and wrapped into barrel structure.
  • The polypeptide chains can turn corner in a number of ways, to go from one β segment or a helix to the next.
  • Not all parts of globular proteins can be conveniently classified as helix, β sheet, or turn.
the information for protein folding figure 6 20
The Information for Protein Folding (Figure 6.20)
  • The information for determining the 3-dimensional structureofa protein is carried entirely in the amino acid sequence of that protein.
  • Native structure is the natural 3-dimensional structure of a protein.
  • Denaturation is a process for the lost of natural structure of a protein, along with many of its specific properties.
  • In a cell, a newly synthesized polypeptide chain will spontaneously fold into the proper conformation.
the thermodynamics of folding
The Thermodynamics of Folding

The overall free energy change on folding must be negative.

  • Conformational Entropy
    • ΔG = ΔH – TΔS
  • Charge-Charge Interactions
    • Salt bridges
  • Internal Hydrogen Bonds (Figure 6.21)
  • van der Waals Interactions (Table 6.3)
  • The Hydrophobic Effect (Table 6.4, Figure 6.22)
slide55
Conclusion: Stability of the folded structure of a globular protein depends on the interplay of three factors.
  • The unfavorable conformational entropy change, which favors random chains instead.
  • The favorable enthalpy contribution arising from intramolecular side group interactions.
  • The favorable entropy change arising from the burying of hydrophobic groups within the molecule.
  • Factor 1 works against folding, whereas 2 and 3 help stabilize folding.
the role of disulfide bonds figure 6 23
The Role of Disulfide Bonds (Figure 6.23)
  • The formation of disulfide bonds between cysteine residues can further stabilize the 3-dimensional structure of proteins, e.g. the BPTI (bovine pancreatic trypsin inhibitor) protein (3 –S-S-- bridges).
  • Formation of the disulfide bonds is depend on the protein primary structure.
  • Disulfide bonds are not essential for correction protein folding, but for contribution to the stability of the structure once it is folded.
  • Proteins found containing disulfide bonds are exported from cells, such as ribonuclease, BPTI and insulin.
  • Site-directed mutagenesis, a powerful method for the test of the effect of changing one or more amino acid residues or adding or removing disulfide bonds.
kinetics of protein folding figure 6 24
Kinetics of Protein Folding (Figure 6.24)
  • Folding of globular proteins from their denatured conformations is a remarkably rapid process.
  • See the rough estimation for the combinations (1050) for a polypeptide chain such as ribonuclease having 124 AA residues, in vitro, the folding time is about 1 minute.
  • From rapid kinetic study, folding takes place through a series of intermediate states, unfolded protein U, nucleation protein, partially folded II, nearly folded IN and the final rearrangement to become native protein F.
slide61
The funneling model for nucleation of proteins proposes that there is not just one but many possible paths from the denatured state to the folded state, and each path leads downhill in energy.
  • The molten globule, a compact structure in which much of the secondary and tertiary folding has occurred, but the internalized hydrophobic residues have not yet formed.
  • There is also off-path state in which some key element is incorrectly folded, and the final folding time will be delayed.
kinetics of disulfide bond formation figure 6 25
Kinetics of Disulfide Bond Formation (Figure 6.25)
  • The process of refolding of a protein from a state in which disulfide bonds have been cleaved is complicated and slower.
  • The refolding process has been studied in great detail by using BPTI.
  • Some disulfide bonds that are not in the native structure are formed in the intermediate stages of the folding.
  • The protein can utilize a number of alternative pathways to fold but ultimately finds both its proper tertiary structure and the correct set of disulfide bonds.
  • It is aided in vivo by enzymatic catalysis of -s-s- bond rearrangement.
chaperonins figure 6 26
Chaperonins (Figure 6.26)
  • Some proteins require special help to achieve proper folding by the aid of special proteins called chaperonins or molecular chaperons.
  • The function of these chaperons is to keep the newly formed protein out of trouble, to avoid improper folding and aggregation.
  • The best studied of all chaperonins is the GroEL-ES complex from E. coli.
  • It provides a shelter for proper folding.
  • ATP is required to drive the process in one direction.
motions within globular protein molecules table 6 5
Motions Within Globular Protein Molecules (Table 6.5)
  • A protein molecule is undergoes continued, rapid fluctuations in its energy, as a consequence of interaction with its environment.
  • The different motions are classified in Table 6.5.
prions protein folding and mad cow disease figure 6 27
Prions - Protein Folding and Mad Cow Disease (Figure 6.27)
  • A class of diseases that is transmitted by a protein and nothing more, and it is against the basic principle that DNA or RNA are the disease information carriers.
  • Bovine spongiform encephalopathy or mad cow disease is the best known of these diseases.
  • The infectious agent is called prion, and the protein is called prion-related protein, or PrP.
  • PrP protein is normally present in many animals including human in a nonpathological form of PrPc (prion-related protein cellular).
  • Under certain circumstances (not yet known), PrPc, can change the conformation to a different structure called PrPsc (or prion-related protein scrapie).
  • The N-terminal portion of PrPsc is partially folded into a β-sheet which wreaks havoc with the nervous system.
  • It can induce conversion of PrPc in the recipient to PrPsc.
prediction of secondary and tertiary protein structure
Prediction of Secondary and Tertiary Protein Structure
  • Can we predict the protein structure? The answer is yes if we could obtain all the necessary information, including the amino acid and understanding the rules of folding.
  • Secondary structure can be predicted fairly well to-date, but not for the tertiary structure.
prediction of secondary structure table 6 6 figure 6 28
Prediction of Secondary Structure (Table 6.6, Figure 6.28)
  • The most successful method for prediction of secondary structure is entirely empirical.
  • From analysis of the known structures of a number of proteins, tables have been complied to show the relative frequency (Pα, Pβ, Pt) with which a particular kind of amino acid residue lies in α-helices, β-sheets or turns.
  • The Chou-Fasman rules for predicts is shown in Table 6.28.
tertiary structure computer simulation of folding
Tertiary structure: Computer simulation of folding
  • Predictions of tertiary structure have proved much more difficult.
  • Attempts to predict the overall three-dimensional structures of small globular proteins depend on their spontaneous folding to seek their minimum free energy.
  • A super-computer is required for protein folding simulation through a large number of various conformation with the rotation about individual bonds.
  • LINUS (Local Independently Nucleated United of Structure), a computer program has made a number of successful predictions.
quaternary structure of proteins
Quaternary Structure of Proteins
  • Many proteins exist in the cell as specific aggregates of two or more folded polypeptide chains, or subunits.
  • Two kinds of quaternary structures: both are multi-subunit proteins.
      • Homotypic: association between identical or nearly identical polypeptide chains.
      • Heterotypic: interactions between subunits of very different structures.
multisubunit proteins homotypic protein protein interaction
Multisubunit proteins: homotypic protein-protein interaction
  • Multisubunit proteins have the same stabilizing forces of tertiary structure, salt bridges, hydrogen bonding, van der Waals forces, hydrophobic interaction, and also the disulfide bonding.
  • Each polypeptide chain is an asymmetric unit in the aggregate, and the overall quaternary structure has a wide variety of symmetries.
  • Heterologous interaction is that interacting groups lie in entirely different regions of the subunit (Fig. 6.30a).
  • Fig. 6.31 for two biological examples.
slide82
Point-group symmetry: involve a define number of subunits arranged about one or more axes of symmetry (Fig. 30b-g).
  • Isologus interaction means that two identical interactions occur, symmetrically placed about the dyad axis, and will be found when 2-fold axes are present (Fig. 6.32).
  • Some dimers do not have 2-fold symmetry (Fig. 6.33).
hetrotypic protein protein interaction
Hetrotypic protein-protein interaction
  • Interaction of BPTI with trypsin (Fig. 6.34).