Lecture 7: Protein structure and function

Question 1

Protein structure is described by four levels of ‘complexity’. What are these four levels?

Answer 1

• Primary structure (1 ̊)
• Secondary structure (2 ̊)
• Tertiary structure (3 ̊)
• Quaternary structure (4 ̊)

Question 2

Primary structure (1 ̊)

Answer 2

• Linear amino acid sequence of the protein

Question 3

Secondary structure (2 ̊)

Answer 3

• Short regions of the peptide backbone fold into individual 3D structures that compose the
  tertiary structure

Question 4

• Tertiary structure (3 ̊)

Answer 4

• The fully-folded, 3D structure of the protein

Question 5

Quaternary structure (4 ̊)

Answer 5

• In many cases, multiple individual proteins combine into a larger, mega-structure

Question 6

The primary structure contains the raw sequence of ___ of the protein

Answer 6

amino acid residues

Question 7

What does the primary structure dteermine?

Answer 7

How the polypeptide folds

Question 8

T/F: Sometimes, a change in single amino acid change have a large effect on protein function

Answer 8

True

Question 9

Mutual Sequence Alignment (MSA)

Answer 9

• Compare primary sequences of different proteins
• Mutant protein vs. wild type
• Homologous proteins from different organisms
• Can also compare nucleotide sequences

Question 10

In MSA, what indicates that all proteins have the same amino acid residue at that position?

Answer 10

asterisk (*)

Question 11

Secondary structures are the _____ that combine into a _________

Answer 11

“building block”
tertiary structure

Question 12

What is the final shape/conformation of a protein?

Answer 12

Tertiary structure

Question 13

Tertiary structures have multiple sub-structures that have distinct shapes called

Answer 13

secondary structures

Question 14

T/F: Proteins fold from primary structure to the secondary structure and then the final tertiary structure.

Answer 14

False, proteins do not fold first
into secondary structures followed by tertiary
* They fold directly from primary structure into tertiary structure
* Secondary structures do not occur
independently

Question 15

Example: Flt3 is a signal transduction protein in human. It is made of single peptide backbone that folds into:

Answer 15

• Four α-helices bundled together
• One β-sheet made of two polypeptide backbones
• Many linkers connecting the α-helices and β-sheets
• Three disulfide bonds holding the linkers together

Question 16

Secondary structure is the folding of the

Answer 16

peptide backbone
* Primary sequence (= sequence of side chains) determines what type of secondary structure that backbone becomes
* However, side chains do not directly stabilize the secondary structure by forming bonds etc.

Question 17

_____ between the backbone atoms
hold the secondary structure together

Answer 17

Hydrogen bonds

Question 18

Two major secondary structures discussed

Answer 18

α-helix and β-sheet are the two major secondary structures, although other types exist

Question 19

α-helix general structure

Answer 19

• Single-stranded, right hand helix
• 0.54 nm per turn
• 3.6 amino acid residues per turn
• Each amino acid residue
  corresponds to a 100° turn
• Peptide backbone is held
  together by hydrogen bonds that
  form within the same backbone

Question 20

α-helix hydrogen bond pattern

Answer 20

• Carboxyl oxygen of aa(N) forms hydrogen bonds with N-H group of aa(N+4)
• N = location of each amino acid residue
• For example, carboxyl oxygen of
  aa1 forms hydrogen bonds with
  N-H group of aa5
• Pattern continues throughout
  the length of α-helix, stabilizing
  the backbone

Question 21

Do the amino acid residues in the α-helix only form one hydrogen bond each?

Answer 21

• Another example. For aa5
• Its carboxyl oxygen is hydrogen
  bonding with NH of aa9
• At the same time, its NH is
  hydrogen bonding with carboxyl
  oxygen of aa1
• Amino acid residues in the
  middle of the α-helix form two
  hydrogen bonds, one above,
  one below

Question 22

Side chains within α-helix

Answer 22

• Polypeptide backbone of the α-helix
  forms a right-handed helix
• Side chain groups point outwards
  from the center of the helix
• Side chain groups do not interact
  with other side chain groups within
  the same helix
• They interact with side chains from other secondary structures in the protein, or with other molecules in the environment

Question 23

What represents the placement of amino acid residues within a α-helix

Answer 23

• Because they are part of a helical
  structure, side chain groups in an α-helix gets placed in a circular pattern
• Helical wheel represents their placement
• Once it folds into an α-helix, similar amino acids gets placed on the same side of the helix
• Polar side of the helix can interact with water
• Hydrophobic side of the helix can interact with other hydrophobic surfaces

Question 24

Amphipathic α-helix

Answer 24

• Amphipathic α-helix have both
  hydrophobic and hydrophilic surfaces
• Sometimes, two amphipathic α-helices wrap around each other
• Their hydrophobic surfaces interact to form a hydrophobic core

Question 25

T/F: all α-helices are amphipathic

Answer 25

False, not all α-helices are amphipathic * Some are completely polar and others are completely nonpolar * Composition depends on the function of the α-helix * For example, a completely nonpolar α-helix can embed itself into the phospholipid bilayer

Question 26

β-sheet general structure

Answer 26

* Multiple polypeptide backbones aligning side-by-side to form a paper-like surface * When viewed from the side, the surface is slightly folded up-and-down ('pleated') * 0.7 nm per 'fold' * ~2 amino acid residues per 'fold' * Cα is located on the top edge and the bottom edge of the 'fold'

Question 27

β-sheet side chains

Answer 27

* Side chain groups stick upwards and downwards in an alternating pattern, perpendicular to the surface

Question 28

β-sheet hydrogen bond formation

Answer 28

* Peptide backbones are held together by hydrogen bonds that form between different backbones * Two amino acid residues from adjacent backbones align * Their carboxyl oxygens and NH groups form hydrogen bonds

Question 29

β-sheet hydrogen bond pattern

Answer 29

* β-sheet has an alternating hydrogen bond pattern * When amino acid pair 1 forms a hydrogen bond, 'pair 2' (beside pair 1) does not form a bond * Amino acid residues of 'pair 2' are forming hydrogen-bonding pair with other peptide backbones * Pattern continues. Pair 3 forms a bond, 'pair 4' does not.

Question 30

Overall shape of the β-sheet

Answer 30

* The plane of the β-sheet is not completely flat – it's twisted

Question 31

Different types of β-sheets

Answer 31

* Peptide backbones in β-sheets can be aligned in two ways * Antiparallel * Parallel

Question 32

Parallel β-sheets

Answer 32

* Parallel β-sheet has its peptide backbones align in a parallel direction * Peptide backbones of parallel β-sheet are held by hydrogen bonds (similarly to antiparallel β-sheet) * However, the hydrogen bond patterns for parallel β-sheets are slightly different

Question 33

Formation of a β-sheet

Answer 33

* Regions of peptide backbones that form a β-sheet do not need to be close to each other in the primary sequence * Two 'distant' areas of peptide backbone can be brought close together in the tertiary structure of the protein * This principle applies to other structures of the folded protein * Distant cysteine residues coming together to form disulfide bonds, etc.

Question 34

Tertiary structure is the

Answer 34

final, 3D conformation of a protein * Individual secondary structures are clustered together in the tertiary structure

Question 35

Examples of tertiary structures

Answer 35

A. Purely α-helix B. Mixed α-helix and β-sheet C. Purely β-sheets

Question 36

Protein domain

Answer 36

a local region of the peptide backbone that folds (somewhat) independently from other regions of the backbone.

Question 37

Many proteins have multiple ___

Answer 37

protein domains

Question 38

Each protein domain belongs to the same

Answer 38

peptide backbone, connected by linker regions

Question 39

Example: Translation factor EF-Tu has three domains

Answer 39

Single peptide backbone folds into three domains * Each domain consist of multiple secondary structures * Three domains combine their activities to give EF-Tu its overall function

Question 40

Individual domains in a multi domain protein may have

Answer 40

their own, independent function * Their functions combine to give the protein its overall activity

Question 41

T/F: A lot of domains can be physically separated from each other and still retain its folding and function

Answer 41

True

Question 42

Domains from different proteins can fuse together to ____

Answer 42

give rise to a novel multi-domain protein

Question 43

Domain shuffling

Answer 43

* Protein domains act like modules that can be put together in different combinations to generate new machines * Nature has taken advantage of this to generate new proteins using domains from already-existing ones * Domain shuffling enables nature to quickly assemble novel proteins without evolving it from scratch

Question 44

Quaternary structure, general information slide

Answer 44

* Multiple individual proteins can combine to form a larger, mega- structure * Individual proteins that constitute the quaternary structure are called subunits

Question 45

Are quaternary structures and proteins domains the same/

Answer 45

* This is different from protein domains * Domains are different regions within the same protein that folds independently * Quaternary structure are made of multiple different proteins

Question 46

Example of a quaternary structure (Hint present in red blood cells)

Answer 46

Human hemoglobin * Heterotetramer composed of two α subunits and two β subunits * Each subunit hold one heme molecule

Question 47

Formation of quaternary structures

Answer 47

* Proteins that form a quaternary structure have surfaces that are shaped to match each other like a lock and a key * Multiple non-covalent bonds form between matching surfaces, allowing them to associate strongly * Disulfide bonds can also hold subunits of quaternary structures

Question 48

Quaternary structure nomenclature, composition

Answer 48

* Composition * 'Homo-' if all the subunits are the same protein * 'Hetero-' if the unit contains at least two species of proteins

Question 49

Quaternary structure nomenclature, number of subunits

Answer 49

* Monomer = 1 protein, by itself * Dimer = 2 proteins forming a unit * Trimer = 3 * Tetramer = 4 * Pentamer = 5 * Hexamer = 6 * Octamer = 8 * etc.

Question 50

DNA binding proteins

Answer 50

* Many proteins bind to specific DNA sequences * They use 'fingers' to reach into the major groove of dsDNA to directly contact the nitrogenous bases * Many DNA binding proteins bind to their target as a dimer * Exceptions exist, of course

Question 51

Example: Pap1 is DNA binding protein belonging to the 'basic region leucine zipper' (bZIP) family