1

Cell Biology & Molecular Biology of The Cell

LecturerDr. Kamal E. M. Elkahlout,

Assistant Professor of BiotechnnolgyLecture 2

Protein Structure and FunctionIntroduction 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)

3

4

All amino acids have the samegeneral structure

5

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

6

7

Basic amino acids have a positivecharge at pH 7.0

8

Acidic amino acids have a negativecharge at pH 7

9

4 of the hydrophilic amino acids arepolar, but uncharged

10

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

11

Selenocysteine is the 21stgenetic 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.

14

15

16

Polypeptide chains are flexible, butconformationally restricted

17

The shape of proteins is determinedthrough 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 playa 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

20

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

22

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. 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. 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

28

29

β-sheets are found in the core ofmany 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 sheetWhere do the side chains go?

31

β-sheets can be in either a parallelor 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. 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

36

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. 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 3-21. 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 3-22. A protein molecule containing multiple copies of a singleprotein 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 theassembly 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.

46

47

Figure 3-38. 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 3-57. Positive regulation caused by conformational couplingbetween two distant binding sites. In this example, both glucose and moleculeX bind best to the closed conformation of a protein with two domains. Becauseboth 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 proteinfunction?

• 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

54

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

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 dephosphorylatedform 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

64

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

67

68

Different graphical representations ofthe same protein

69

Different graphical representations ofthe same protein