1. Cell Biology & Molecular Biology of The Cell
Lecturer
Dr. Kamal E. M. Elkahlout,
Assistant Professor of Biotechnnolgy
Lecture 2
Protein Structure and Function
Introduction to biotechnology registration for Biotech MSc

2. Protein Central Dogma
Proteins are large molecules that are formed as single, unbranched chains of amino acid monomers
– But, proteins can be turned into branched structures by ubiquitin and other ubiquitin-like molecules
There are 20 different amino acids commonly found in proteins
• A protein’s amino acid sequence determines its three dimensional structure (conformation)
– Well, sort of….
A protein’s 3-dimensional structure determines its chemical function(s)
– (along with a whole lot of different post-translational modifications that can alter parts of its structure and change its functions)

4. All amino acids have the same general structure

5. Fig. 2-14: The 20 common
amino acids found in proteins.

7. Basic amino acids have a positive charge at pH 7.0

8. Acidic amino acids have a negative charge at pH 7

9. 4 of the hydrophilic amino acids are polar, but uncharged

10. The remaining amino acids have hydrophobic and “special” functional groups

11. Selenocysteine is the 21st genetic encoded amino acid

12. Amino acids are linked by an amide linkage, called a peptide bond, to form polypeptide chains

13. Peptide bonds and the α carbon atoms form the linear backbone of proteins, which is a regular, repeating unit
The functional groups of amino acids form “side chains” that are connected to the backbone.

16. Polypeptide chains are flexible, but conformationally restricted

17. The shape of proteins is determined through 4 levels of structure
Primary: the linear sequence of amino acids
Secondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet)
Tertiary: the overall, three-dimensional arrangement of the polypeptide chain
Quaternary: the association of two or more polypeptides into a multi-subunit complex
The final, 3-dimensional, folded structure is generally one in which the free energy of the molecule is minimized

18. Three types of weak, non-covalent bonds also constrain the folding of proteins into their energy minimized 3-D structures

19. Hydrophobic interactions also play a role in determining protein shape
Residues with hydrophobic side chains tend to cluster in the interior of the protein molecule, avoiding contact with water
Polar side chains tend to be arranged on the outsides of proteins in contact with the aqueous medium

21. All of these bonds are about 30-300 times weaker than covalent bonds
So why are they important?
Many weak bonds applied together can produce a large force.
The stability of a protein is determined by the combined strength of many non-covalent bonds

23. Secondary Structure
The α-helix and the β-sheet are two regular folding patterns found in almost all proteins
What produces these structures and why are they so common?
They result from hydrogen bonding between multiple N-H and C=O groups in the backbone.
Side chains are not involved in these structures.

24. The α-helical backbone is a rigid cylinder with the amino acid side chains protruding from its surface

25. ▲ FIGURE 3-2 Structure of a tripeptide
▲ FIGURE 3-2 Structure of a tripeptide. Peptide bonds (yellow) link the amide nitrogen atom (blue) of one amino acid (aa) with the carbonyl carbon atom (gray) of an adjacent one in the linear polymers known as peptides or polypeptides, depending on their length. Proteins are polypeptides that have folded into a defined three-dimensional structure (conformation).
The side chains, or R groups (green), extending from the carbon atoms (black) of the amino acids composing a protein largely determine its properties. At physiological pH values, the terminal amino and carboxyl groups are ionized.

26. ▲ FIGURE 3-3 The helix, a common secondary structure in proteins
▲ FIGURE 3-3 The helix, a common secondary structure in proteins. The polypeptide backbone (red) is folded into a spiral that is held in place by hydrogen bonds between backbone oxygen and hydrogen atoms.
The outer surface of the helix is covered by the side-chain R groups (green).
Side chains protrude from the surface of the cylinder

27. α-helices can form very stable coiled-coil structures through hydrophobic interactions between non-polar side chains

29. β-sheets are found in the core of many proteins
β-sheets are rigid, relatively flat and extended structures that are stabilized by hydrogen bonds between neighboring polypeptide strands

30. Secondary structure: the beta sheet Where do the side chains go?

31. β-sheets can be in either a parallel or antiparallel orientation

32. Most extracellular proteins are stabilized by covalent –S-S- cross links Disulfide bond formation is catalyzed in the ER prior to export

33. Protein domains represent another important unit of organization

34. Figure 3-12. A protein formed from four domains
Figure A protein formed from four domains. In the Src (tyrosine kinase involved in signaling between cells in multicellular animals) protein shown, two of the domains form a protein kinaseenzyme, while the SH2 and SH3 domains (Src homolgy domain2 & 3) perform regulatory functions. (A) A ribbon model, with ATP substrate in red. (B) A spacing-filling model, with ATP substrate in red. Note that the site that binds ATP is positioned at the interface of the two domains that form the kinase.

35. Hierarchical Structure of Proteins
Domains are constructed from different combinations of α-helices and β-sheets at their core
Each combination is called a protein fold

37. Most large multi-domain proteins have evolved by recombination and joining of preexisting domains in new combinations (Domain Shuffling)
Many small molecule binding sites in proteins are created at the surfaces between new combinations of domains

38. Figure 3-18. Domain shuffling
Figure Domain shuffling. An extensive shuffling of blocks of protein sequence (protein domains) has occurred during protein evolution. Those portions of a protein denoted by the same shape and color in this diagram are evolutionarily related.
Serine proteases like chymotrypsin are formed from two domains (brown).
In the three other proteases shown, which are highly regulated and more specialized, these two protease domains are connected to one or more domains homologous to domains found in epidermal growth factor (EGF; green), to a calcium-binding protein (yellow), or to a "kringle“ domain (blue) that contains three internal disulfide bridges.

39. Large proteins often contain more than one polypeptide chain
Binding between two protein surfaces generally involves sets of non-covalent bonds

40. Figure Two identical protein subunits binding together to form a symmetric protein dimer. The Cro repressor protein from bacteriophage lambda binds to DNA to turn off viral genes.
Its two identical subunits bind head-to-head, held together by a combination of hydrophobic forces (blue) and a set of hydrogen bonds (yellow region).

41. Figure A protein molecule containing multiple copies of a single protein subunit. The enzyme neuraminidase (glycoside hydrolase nz, neuraminin acid) exists as a ring of four identical polypeptide chains. The small diagram shows how the repeated use of the same binding interaction forms the structure.

42. Figure 3-23. A protein formed as a symmetric assembly of two different subunits.
Hemoglobin is an abundant protein in red blood cells that contains two copies of a globin and two copies of b globin.
Each of these four polypeptide chains contains a heme molecule (red), which is the site where oxygen (O2) is bound.
Thus, each molecule of hemoglobin in the blood carries four molecules of oxygen.

43. Some globular proteins can form long helical filaments
• Globular proteins fold into a compact, ball-like shape with irregular surfaces
• Example: Actin filaments form in a helical arrangement that can be the length of the cell

44. Proteins can be subunits for the assembly of large structures
enzyme complexes
ribosomes
Proteasomes (large proteases, degrade uneeded damage proteins)
filamentous structures (nuclear lamina)
viruses
membranes

45. Protein Function Some General Principles
All proteins bind to other molecules
Protein binding has a high degree of specificity for its ligands (binding partners)
Ligand specificity and affinity are determined by sets of weak non-covalent bonds and hydrophobic interactions.

47. Figure 3-38. The binding site of a protein
Figure The binding site of a protein. (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can make noncovalent bonds only with certain ligands. (B) A close-up of an actual binding site showing the hydrogen bonds and ionic interactions formed between a protein and its ligand (in this example, cyclic AMP is the bound ligand).

48. Enzymes are highly specific catalysts
Enzymes speed reactions by selectively stabilizing unstable transition states (conformations) of their ligands
This lowers the activation energy of the reaction.

49. The catalytic activities of many enzymes are highly regulated through small molecule binding sites
Allosteric enzymes have two or more binding sites that interact with other molecules
– an active site that recognizes substrates
– a regulatory site that recognizes a regulatory molecule binding of a regulatory molecule at one site on the protein causes a conformational change in the polypeptide that can switch the active site conformation “On” or “Off”.

50. Figure Positive regulation caused by conformational coupling between two distant binding sites. In this example, both glucose and molecule X bind best to the closed conformation of a protein with two domains. Because both glucose and molecule X drive the protein toward its closed conformation, each ligand helps the other to bind. Glucose and molecule X are therefore said to bind cooperatively to the protein.

51. How does a cell regulate protein function?
Cells can regulate the steady state levels of proteins through synthesis or degradation
– Regulate mRNA levels by controlling transcription or mRNA stability
– Control of translation of a protein’s mRNA
– Targeted degradation of a protein through proteolysis
Changing the activity of a protein through conformational changes
Changing the location of a protein by moving it to a different part of the cell

52. Protein structure and function can also be regulated by covalent modifications of exposed residues N-terminal acetylation stabilizes proteins – non-acetylated proteins are degraded rapidly by proteases

53. Acetylation and Deacetylation of Histone Tails Control Transcription Activity
Deacetylation inhibits binding of transcription factors to the TATA box, repressing gene expression
Hyperacetylation of histone N-terminal tails facilitates access of general transcription factors needed for transcription initiation

55. A few of the chemical modifications found on functional groups on internal residues

56. Ubiquitin is one of a family of small proteins that can be covalently linked to the ε- aminogroup of exposed lysine residues
Ubiquitin covalently links its C-terminal Glycine residue to the ε-amino group of lysine through an iso-peptide bond.

57. Polyubiquitination can target proteins for degradation in proteasomes

58. Cells contain a large collection of protein kinases and phosphorylases
Monoubiquitination, or Linkage of Small Ubiquitin-Like Molecules (SUMOS) Can Regulate Protein Structure and Activity
In cells, many changes in protein binding / catalytic functions are driven by phosphorylation
Cells contain a large collection of protein kinases and phosphorylases
What amino acids are phosphorylated?

59. Phosphorylated amino acids

60. Protein phosphorylation and dephosphorylation play a major role in regulating enzyme activity and in driving the regulated assembly and disassembly of protein complexes
Addition of a phosphate group (2 – charges) to a residue can attract + charged side chains, causing major conformation changes
Attached PO4 groups can form structures that can be recognized as binding sites by other proteins.

61. Phosphorylation / dephosphorylation
Protein phosphorylation is reversible and can act as a molecular switch
Dephosphorylation can restore original conformation and activity of the protein
Note: In this case the dephosphorylated
form of the protein is active

62. Other proteins bind and hydrolyze GTP to act as a molecular switch (GTP binding proteins or GTPases)
Actually another form of phosphorylation/ dephosphorylation
GTP binds tightly to protein, usually activating it.
Protein can self-catalyze conversion from GTP to GDP.
Conformational change converts protein to inactive form.

63. Activation of Ras signaling causes cell growth deferentiation and survival

65. GEF guanine exchange facto, GAP GTPase activating factor

66. Phosphoproteins can serve as signal integrators for a molecular switch
Example: Activation of a protein requires the input of multiple signals from different parts of the cell
cdk kinase (cyclin dependent kinase) – involved in cell division

68. Different graphical representations of the same protein

69. Different graphical representations of the same protein