Protein Structures and Functions

Question 1

What do proteins provide?

Answer 1

· Carrier functions-trafficking oxygen, importing and exporting molecules such as glucose or ions
· Metabolic functions- Enzyme cascades producing and utilizing energy glycolysis or TCA cycle
· Form parts of the Cellular machinery-sometimes as parts of ribonucleoprotein complexes such as spliceosomes, ribosomes
· Make up structural scaffold- microtubules, nucleosomes
· Sensing molecules-cell surface receptors and their ligands

Question 2

Why is the orientation of amino acid side chains important?

Answer 2

The side chains form the specific contacts within and between proteins, cofactors and enzyme substrates or ligands.

Question 3

How is the basic structure of an amino acid determined?

Answer 3

By the valency of carbon

amino acid has a tetrahedral arrangement of atoms around a central alpha carbon

Question 4

Describe the basic structure of an amino acid

Answer 4

Linked to the C-alpha carbon are a carboxyl and amino group, a single hydrogen and a variable side chain usually represented by R and a hydrogen group

Question 5

Explain the ionic properties of amino acids

Answer 5

· The carboxylic acid and amine groups of the amino acids are readily ionized.
· This means that the NH2 group can act as a base and acquire a hydrogen becoming an NH3+ ·

Similarly the carboxyl group readily donates a hydrogen acting as an acid becoming COO-

· The various groups of an amino acid are in equilibrium with the surrounding environment

Question 6

Explain the states of amino acids in low, neutral and high pH

Answer 6

· at high pH the carboxyl group donates a hydrogen and becomes negatively charged
at low pH the amine group acquires a hydrogen becoming positively charged

· And At neutral pH the molecule has an overall balanced charge and contains both positively and negatively charged groups and is described as a zwitterion

Question 7

Which amino acid does not have two isomers?

Answer 7

Glycine because the R group is a hydrogen.

Question 8

How does D and L isomers arise?

Answer 8

there are two possible isomers for every amino acidbecause ofthepossibility of forming two different enantiomers (stereoisomers) aroundthecentral carbon atom.

Question 9

What nature is the C-alpha?

Answer 9

chiral, assymetrical

Question 10

Describe the difference between L and D amino acids

Answer 10

· L-amino acids are found in naturally occurring proteins of all living organisms.

D amino acids do exist in nature and can be found in the cell walls of some bacteria but are also sometimes used as therapeutics

Question 11

How do peptide bonds form?

Answer 11

condensation reaction releasing H2O

Question 12

What is a residue?

Answer 12

· Each repeating unit of the polypeptide chain is termed a residue

Question 13

How does polarity arise in amino acids?

Answer 13

Amino (N) and Carboxyl (C) terminus

Question 14

How is the R chain oriented and what is the implication?

Answer 14

· arranged in a trans conformation such that the variable side chain is alternately orientated.

This orientation occupies space with the lowest energetic requirement

Question 15

What is the difference between cis and trans orientation of amino acids energy wise?

Answer 15

· 0.1% peptide bonds have a less energetically favorable cis arrangement- where the side chain is located on the same side

Question 16

How do secondary structures arise?

Answer 16

· They are formed from intra-chain hydrogen bonding ie bonding between different parts of the same chain

Question 17

How do beta pleated sheets arise and how are they connected?

Answer 17

· Held together by intramolecular hydrogen bonds · the chain is folded to form a parallel or anti-parallel arrangement
· The orientation of the strands determines the relative positioning of the groups forming the hydrogen bonds affecting their strength and stability

· the beta strands are connected either by longer omega loops or short turns of the polypeptide chain.

Question 18

Explain the difference between antiparallel and parallel arrangement of beta strands

Answer 18

Anti-parallel arrangement of beta strands produces a beta sheet with greater stability and strength because hydrogen bonds in anti-parallel beta sheet are linear

Question 19

How do beta bends/reverse turns form?

Answer 19

· involve four amino acid residues with a hydrogen bond between the C=O group of the first residue and the N–H group of the fourth.

Type I and type II β bends differ in respect of the torsion angles for the residues.

Question 20

How do beta loops form?

Answer 20

· Non-regular motif, consisting of a loop of six or more amino acid residues and any amino acid sequence.

2. Residues that make up the beginning and end of the loop are close together

· Turns and loops lie on the surfaces of proteins and thus often participate in interactions between proteins and other molecules.
· Recognition role of proteins, such as the recognition of specific antigens by antibodies.

Question 21

Explain how alpha helices form(3)

Answer 21

· Hydrogen bonds are 4 residues apart
· Alpha helices form a right-handed helix with 3.6 residues per turn and approximately 0.54 nm per turn · The side chains form the specific contacts within and between proteins , cofactors and enzyme substrates or ligands.

Question 22

How does the tertiary strcuture arise?

Answer 22

· Combining secondary structures such as alpha helices, beta sheets and beta turns

these bonds and forces are acting between the same chain in tertiary structure

Question 23

Give an example of a protein in its tertiary structure in a membrane

Answer 23

· the seven transmembrane domain of the thyroid stimulating hormone receptor or the CXCR4 chemokine receptor
· The alpha helices in each case span the membrane and anchor the structure in the membranes

Question 24

How are quaternary structures formed?

Answer 24

· combining two or more folded polypeptide chains, the same bonds and forces influence quaternary structure
· however a key difference is that these bonds and forces are acting between different polypeptide chains

Question 25

Give an example of protein at a quartenary structure

Answer 25

·       hemoglobin Quaternary structure is formed by combining four chains  2 Hbα and 2 Hbβ polypeptides

Question 26

Explain the various sizes and shapes of proteins with different examples

Answer 26

·       The size and complexity of functional proteins vary hugely Haemoglobin comprises of 4 chains , but insulin has just 2, Glutamate synthase may have between 8 and 12 depending on the organism, ·       ribosomes are ribonucleoproteins comprising of both RNA and large numbers of multi-chain proteins. 

Question 27

What does the formation of structure and the ability for a protein to function depend on?

Answer 27

Often dependent on cofactors ·       Cofactors may also be needed for the structural integrity and correct folding of the protein. For example, in hemoglobin both haem and iron are present, in insulin zinc is critical for its structure and function. 

Question 28

What are cofactors?

Answer 28

A cofactor is a non-protein chemical compound that is required for the protein's biological activity They are active centres for NAD and FAD

Question 29

Are cofactors limited to tertiary structures?

Answer 29

No, they can be found with quaternary structures

Question 30

Describe the orientation of water solube proteins

Answer 30

·       Hydrophilic residues are mostly on the external surface. ·       Hydrophobic residues are usually buried inside the protein ·       Not all water soluble proteins are globular and some form filaments eg actin, tubulin or cortexillin but in all these the hydrophilic amino acids face outside

Question 31

Describe the orientation of proteins in non-aqueous environments and give an example

Answer 31

·       hydrophilic residues may be buried internally. ·       the Hydrophobic residues of membrane bound receptors may anchor a protein in the hydrophobic environment of the membrane eg the CXCR4 chemokine receptor

Question 32

How is folding of the polypeptide chain determined?

Answer 32

*        the amino acid sequences *        the molecular structure and properties of its amino acids *        the molecular environment (ligands, cofactors, solvents & salts)

Question 33

Give examples of charged amino acids

Answer 33

aspartic acid, glutamic acid, lysine and arginine

Question 34

Describe charged amino acids

Answer 34

1. negatively and positively charged and 2. charged groups within the amino acids are either carboxylic acids or amines  •       charged amino acids are also polar in nature but are sufficiently different from the polar amino acids

Question 35

Give examples of non-polar or hydrophobic amino acids

Answer 35

alanine, valine, leucine, isoleucine Special case: methionine 

Question 36

Describe sulfur contaning amino acids and give examples

Answer 36

sulphur containing amino acid is cysteine so whilst having a group containing both make some sense,

Question 37

Give examples of polar amino acids

Answer 37

Serine, threonine •       Histidine is also sometimes considered as a charged amino acid Tyrosine histidine and tryptophan are in a sub group the polar aromatic amino acids

Question 38

What is the difference between polar amino acids and the charged amino acids?

Answer 38

Polar amino acids contain nitrogen and oxygen atoms as part of carbonyl or secondary amide groups rather than carboxyl or amine groups

Question 39

Why are glycines found in turns or loops?

Answer 39

It is small and provides more freedom of movement in the chain.

Question 40

How does peptide bonds affect protein folding?

Answer 40

·       The peptide bond imparts restrictions on the folding of the chain •       flat planar structure that has a fixed arrangement because of delocalised electrons associated with the carbonyl group and amide groups. t the peptide bond behaves as if it were a double bond but is not. *        However, the rest of the chain has rotational freedom around the bonds of the c alpha atom ie the c alpha- c prime and c alpha and the nitrogen. The peptide bond itself is rigid. *        And this provides the freedom of movement to dynamically form the complexity of secondary, tertiary and quaternary structure eg alpha helicies. 

Question 41

What can happen to amino acids with large side chains?

Answer 41

•       Amino acids with large side chains may be restricted in their rotation

Question 42

What is the intrinsic state of proteins?

Answer 42

·       All molecules have an intrinsic energetic state and this energetic state determines its conformation and minimization of this drives changes toward a minimum energetic state

Question 43

What determines arrangement of atoms?

Answer 43

•       The minimisation of this energetic state (the free energy of a molecule “G”) determines the most favourable arrangement of the atoms (conformation) 

Question 44

What is ∆G?

Answer 44

•       the change in free energy upon folding is called ∆G

Question 45

Is ∆G negative or positive upon simultaneous fold?

Answer 45

It is negative

Question 46

What is the free energy of structure affected by?

Answer 46

·       by the environment and the other molecules that it interacts with •       for example aqueous vs lipid environment, •       ligands, cofactors or ions etc •       and changes in that environment as a result of binding a ligand or cofactor can result in conformational changes and a different structure and thus protein structure is dynamic

Question 47

What is the difference between covalent and non-covalent bonds?

Answer 47

with Non- covalent bonds being weakernbeing only 1/20th the strength of covalent bonds •       However the number of non-covalent bonds that contribute to the overall structure of a protein is much greater, and are numerous by comparison

Question 48

Recall the different types of non-covalent bonds

Answer 48

Charge or electrostatic attractions  van der waals attractions  hydrophobic interactions

Question 49

Describe charge or electrostatic attractions of non-covalent bonds

Answer 49

·       falls off exponentially as distance increases, affected by electrostatic environment (aqueous environment) ·       Hydrogen bonds (transient bonds similar in some respects to covalent bonds) 

Question 50

Describe van der waals attraction

Answer 50

Dipole- weak forces occur between two atoms ·       determined by their fluctuating charge ·       attraction at a close distance is balanced by repulsion due to proximity that is determined by the Van der Waals radius of an atom,

Question 51

Describe hydrophobic interactions and how they minimise disruption of water network

Answer 51

*        (water is a polar molecule) hydrophobic interactions minimise disruption of water network – ie the fourth weak force *        water is a polar molecule that forms a network of polar interactions, disruption of this network comes at an energetic cost *        a protein will adopt a structure that minimises that network *        for example forming a globular structure with hydrophilic groups on its external surface in an aqueous environment

Question 52

How are disulphide bonds formed?

Answer 52

*        These bonds are formed from an oxidative reaction between cysteines that come into proximity because of folding *        and can form between cysteines on the same polypeptide chain (tertiary structure) or between different polypeptide chains (quaternary structure)

Question 53

What is the role of covalent bonds in a folded structure?

Answer 53

Covalent bonds involved in the formation and stabilization of a folded structure

Question 54

Recall diseases related to protein misfolding

Answer 54

*        Huntingtin Htt (Huntington’s) *        Amyloid-beta Ab (Alzheimer’s) beta-amlyloid plagues form *        prion protein (PrPSc) Creutzfeldt–Jakob disease and variant CJD *        alpha-synuclein (Parkinson’s disease) *        Serum amyloid A (AA amyloidosis) *        islet amyloid polypeptide IAPP (Type 2 Diabetes)

Question 55

Which misfolding form aggregates?

Answer 55

Secondly mis-folded proteins 

Question 56

How does cystic fibrosis arise from protein misfolding?

Answer 56

mis-folded proteins result in cellular processing that lead to their degradation. Lack of protein contributes to disease. 

Question 57

How does Cystic fibrosis arise?

Answer 57

·         the most common mutation is a deletion of Phenylalanine at residue 508 of the cystic fibrosis transmembrane conductance regulator (CFTR)- a single codon deletion ·         CFTR regulates chloride ions crossing the cellular membrane ·          DF508del leads to mis-folding of the protein whilst it is still in the ER ·         This is recognised by the cellular machinery that identifies and processes misfolded protein ·         This results in ubiquitination, trafficking to the proteasome and degradation ·         An example of somatic deletion

Question 58

Why does mis-folding occur? (6)

Answer 58

* somatic mutations in the gene sequence leading to the production of a protein unable to adopt the native folding * errors in transcription or translation leading to the production of modified proteins unable to properly fold * failure of the folding machinery * mistakes of the post-translational modifications or in trafficking of proteins *  structural modification produced by environmental changes * induction protein mis-folding by seeding and cross-seeding by other proteins

Question 59

How does Alzheimers arise? (8)

Answer 59

1. b-Amyloid (Ab) is a small protein released because of proteolysis from a larger transmembrane protein known as Amyloid Precursor Protein APP 2. beta amyloid is normally lying within the membrane 3.  When it accumulates in isolation of the rest of the protein it misfolds and then forms beta sheets which in turn form stacked multimers.  4.  Flat planar monomeric sheets form, those in turn aggregate to form stacked sheets of oligomers 5.  The oligomers  extend to form protofibrils that further aggregate forming an amyloid fibrils 6. Protofibrils interdigitate with those of another sheet forming amyloid fibril 7. The aggregation of b-amyloid in the brains of Alzheimer’s patients is a feature of the pathology of the disease .  8. These interfere with the workings of the synapse, particularly in the hippocampus. Gradually, higher order insoluble, aggregates form, deposited in plaques, damaging the neuronal cells of brain.

Question 60

What kind of modification occurs in Alzheimer's?

Answer 60

an example of a post-translational modification of the protein resulting in proteolysis leading to disease pathology

Question 61

How does induced protein misfolding occur?

Answer 61

Mis-folded proteins(prion proteins) that interact with other normal proteins  ·       Through this interaction they induce mis-folding of the normal protein and polymerisation ·       by seeding and cross-seeding by mis-folded ·       Oligomers form fibrils of mis-folded protein- induction is a change in energy that represents a lower energetic state in the oligomeric structure than in the correctly folded molecule in isolation 

Question 62

Why are aggregate structure more stable?

Answer 62

·       This process is reliant upon the concept of energy minimisation ∆G •       It is a dynamic process brought about by the interaction of  molecules resulting in a more stable aggregated structure 

Question 63

What is a protein sequence domain?

Answer 63

a pattern of amino acids that are found in related genes or proteins

Question 64

What are motifs and domains?

Answer 64

*        Simple secondary structures (folds) combine to form structural motifs or larger functional domains *        Such units of protein structure are commonly found and are conserved across functionally related proteins but may not be related in sequence nor direct evolutionary pathway

Question 65

What is a motif?

Answer 65

*        a minimum arrangement of independently forming secondary structures combining recognisable folds (arrangements) across many different proteins *        A combination of two or more secondary structures to form a recognisable localised folded arrangement of structure *        Motifs are organised or combined into larger structural and functional domains

Question 66

How are motifs coded for?

Answer 66

•       They are coded by segments of genes; they are modular in nature and relate back to a heritable unit of genetic structure for example functional domains often but not always relate to a single exon within a gene

Question 67

What is a domain?

Answer 67

a conserved part of the protein sequence that can function independently to the rest of the protein chain. They are a region that has a specific function •       a more complex structure at the tertiary or quaternary level, often involving interaction between distant parts of a protein or motifs

Question 68

What is a functional domain?

Answer 68

•       typically larger and may or may not be a contiguous segments of a single polypeptide chain •       A functional would comprise of multiple modular domains each with a different function  

Question 69

Give an example of a functional domain 

Answer 69

•       SRC (pronounced SARK) kinase would have multiple domains, both kinase and regulatory domains each with different functions, the SH4 domain is unique to but the others are modular units combined elsewhere in different proteins

Question 70

How many structural motifs exist?

Answer 70

1400-1500 different structural motifs (folds) 

Question 71

What motifs are found in the TATA box binding proteins?(3)

Answer 71

a beta-alpha-beta. the polypeptide chain of a single beta strand associated with the beta sheet then connects directly to the alpha helix of the motif, the polypeptide chain then forms a series of four antiparallel beta strands making up the remainder of the beta sheet on the right, followed by the longer alpha helix sitting on top of the beta sheet.

Question 72

What do beta-alpha-beta motifs do to DNA through TATA box binding proteins?

Answer 72

The overall structure comprises of two such motifs together they bind to and distort the DNA recruiting other proteins to form the initiation complex allowing transcription to start

Question 73

What are the two common motifs?

Answer 73

 the EF hand and the Greek Key

Question 74

What is the EF hand?

Answer 74

EF hand is a calcium binding motif found in for example in Calmodulin & Troponin-C , it combines with a metal ion calcium shown. There are 2 EF hands at either end of an alpha helix going.

Question 75

What is the Calmodulin in EF hand?

Answer 75

Calmodulin is part of the mechanism for sensing intracellular calcium levels and calcium binding to the EF hand results in a conformational change in the protein

Question 76

What is the Greek Key motif?

Answer 76

consists of a specific arrangement of beta strands within an antiparallel beta sheet, but is one motif that is so common it isn’t generally associated with a specific function.

Question 77

What is the beta barrel motif?

Answer 77

arrangement of beta strands wrapped around to form a circular tunnel that looks somewhat like a wicker basket,  a similar to alpha/beta barrel which is also surrounded by alpha helices These motifs are also associated with a diverse set of functions

Question 78

What is the beta-alpha-beta motif?

Answer 78

Parallel strands of a beta sheet are interlinked with a alpha helix

Question 79

Where in the DNA are DNA binding motifs inserted?

Answer 79

inserted into the major groove of DNA in a sequence specific manner

Question 80

Recall the four DNA binding motifs studied

Answer 80

·       Helix loop helix – eg Max & Mad also Ca2+ binding ·       Helix turn helix –eg Cro, tryptophan, & lac repressors ·       Leucine Zipper – eg GCN4 ·       Zinc Finger – eg  hormone receptors

Question 81

What is the role of functional domains?

Answer 81

anchoring the protein in the membrane

Question 82

What is the most common form of fucntional domains?

Answer 82

Bundles of alpha helices less commonly lone helices or a bundle of beta sheets

Question 83

Describe an example of a functional domain in the membrane

Answer 83

The 7-transmembrane domain arrangement of alpha helices is common found in rhodopsin, TSHr, many pharmacological receptors and receptors for some polypeptide hormones

Question 84

Describe an example of domain shuffling

Answer 84

Mammalian phospholipase C, this contains 4 different recognisable domains. Each of these is found individually found in other proteins troponin C , bacterial phospholipase C etc   Since each of these are linked to a segment of the gene

Question 85

How is haemoglobin and myoglobin examples of functional domains?

Answer 85

Each chain of haemoglobin has a tertiary structure very similar to that of the single myoglobin chain, strongly suggesting evolution from a common ancestral O2- binding polypeptide

Question 86

Why are alpha helices referred to as recognition helix?

Answer 86

An alpha helix can fit within the major groove of DNA – this structure forms the specific contacts with the DNA bases

Question 87

What is common between the binding of the motifs to DNA?

Answer 87

only in the dimeric form and the Helix loop Helix typical of this

Question 88

Describe the helix-loop-helix(6)

Answer 88

1. One of the largest families of dimerizing transcription factors.  2. bHLH proteins bind to a consensus sequence called the E box 3. Exists as  hetero (different monomers) and homodimers (identical monomers) 4. The central portion of the HTH is made from overlapping helices that form a structure enabling dimerisation, this dimerization domain forms a structure that resembles a twisted rope. 5. The terminal part of the lower opposing helices contain basic amino acids that interact with the major groove of the DNA – giving rise to the b/HLH functional domain.  6. The central part hold the recognition helices in place

Question 89

Give examples of Helix-loop-Helix 

Answer 89

MYOD, MYF5, MAX, TCF4- master gene regulators

Question 90

Describe the Leucine Zipper motif(4)

Answer 90

1. Formed from 2 contiguous alpha helices and like the HLH, is a dimeric protein formed from two polypeptide chains. 2. The dimers again “zip” together in the top “stalk” to form a short “coiled-coil” 3. The coil is held together by hydrophobic interactions down opposing sides of the helix 4. As in the b/HLH basic amino acids dominate the lower part of the helix and since they are basic repel each other opening the terminal ends like the jaws of a clamp and interact with the DNA major groove of the negatively charged DNA

Question 91

Why is heteromonomers and homodimers a key characteristic in helix-loop-helix motifs?

Answer 91

•      allow different dimers to regulate different genes as a consequence of each monomer that comprises the dimer recognising  a different sequence in the DNA

Question 92

Give examples of Leucine Zipper motif

Answer 92

"cFon, cJun

Question 93

How are motifs involved in DNA transciption?

Answer 93

These combine to form domains that have specific functions such as DNA binding, dimerization, and other regulatory functions These motifs are conserved across all phyla

Question 94

Describe helix-turn-helix and their interaction with DNA

Answer 94

two short helices orientated at right angles to each other & connected by a “turn” but this is not a beta turn 2. The motif is found in both prokaryotic and eukaryotic DNA binding proteins for example the CRO and lambda repressors, & homeobox proteins  CRO and Lambda are homodimers, 3. CRO therefore recognises a palindromic sequence and by binding DNA represses transcription 4. Only the recognition helix interacts with the nucleotide sequence itself 5. the helix turn helix locates the recognition helix within the major groove

Question 95

Describe zinc finger motifs

Answer 95

An alpha helix and a beta sheet held together by non-covalent interactions with zinc 2. The alpha helix of each motif interacts with the major groove of DNA and recognises a specific DNA sequence.   3. different parts of the dimer contribute to the DNA binding domain in addition to each of the motifs, 4.allowing regulation of transcription

Question 96

Recall examples of zinc finger homone receptors

Answer 96

Glucocorticoid, Mineralcorticoid oestrogen, progesterone, as well as Vit D receptors