Proteins

Question 1

Peptide Bonds

•___ reaction – release of ____ during bond formation

• Peptide unit is ___ and ____
• Assumes a ____ configuration. (Which bond)
• Free rotation occurs around the ____ & _____bonds adjacent to the peptide bond.

_____ character for ____ bond

There is resonance with ____ double bond

Peptide bond is hardser to break then regular single bond

Answer 1

•Condensation reaction – release of water during bond formation

• Peptide unit is rigid and planar.
• Assumes a trans configuration. (C=O and NH)
• Free rotation occurs around the C-Ca1 and Ca2-N bonds adjacent to the peptide bond.

Double bond character for CN bond

There is resonance with C=O double bond

Peptide bond is hardser to break then regular single bond

Question 2

Properties of Peptide Bonds

Peptide bonds are ____

___ form is favored (CO and NH). Less ___ ____ (bw ____ ____) occurs in the ___ than in the ____ form

Peptide bond is ____, however, bonds between the a-carbon and the ____ ____ (___) and ____ atom (___) are able to rotate.
Rotation about these bonds can be defined by the___ ____.
The rotation about these bonds allow proteins to ____in different ways.

Answer 2

Peptide bonds are planar

Trans form is favored (CO and NH). Less steric collisions (bw side groups)occurs in the Trans than in the cis form

Peptide bond is rigid, however, bonds between the a-carbon and the carbonyl carbon (Psi) and nitrogen atom (Phi) are able to rotate. Rotation about these bonds can be defined by the dihedral angles. The rotation about these bonds allow proteins to fold in different ways.

Question 3

Levels of Structure with in a Protein

The structure of a protein is determined by the

Answer 3

The structure of a protein is determined by the amino acid sequence.

Question 4

Primary Structure of a Protein

Answer 4

•The linear sequence of amino acids of a polypeptide chain.

Question 5

Assess the Charge on a Small Peptide at a given pH
•The charge of an amino acid at given ____ is a function of the ____ of the dissociation of protons by the ___ ____ and ____ groups.
•The isoelectric point is the
•The charge of a polypeptide chain can be determine by considering the _________of amino acids,

Answer 5

• The charge of an amino acid at given pH is a function of the pKa of the dissociation of protons by the carboxyl, amino and the side chain groups.
• The isoelectric point is the pH at which the net charge of the molecule is zero.
• The charge of a polypeptide chain can be determine by considering the pKa of the side groups of amino acids, the amino and carboxy groups at the N and C termini.

Question 6

pKa Values of Amino Acid Side Chains

Answer 6

Remember the amino group and the carboxyl group at the N and C termini of the polypeptide chain have pKa values of ~ 9 and ~2 respectively.

Asp (D)

Glu (E)

His (H)

Cys (C)

Tyr (Y)

Lys (K)

Arg (R)

Question 7

Calculate the charge on the polypeptide chain at pH=7.4

Answer 7

At low pH all the groups (Acidic and basic groups) are protonated.

As the pH increases the side groups will dissociate in order of their increasing pKas.

Negative charges will be contributed by the carboxyl groups of Glu, the two Asp and the carboxyl group of the C terminus. (Total negative charge will be -4)

The positive charge will be contributed by the amino groups of Lys and N-terminal amino group. (Total positive charge will be +2)

Ala, Phe and Pro do not contribute any charge to the peptide (non-polar and hydrophobic groups)

Hence total charge on the peptide will be -2 (-4 + +2)

pH< pka Protonated

pH > pka Deprotonated

Question 8

Secondary Structures of Proteins

•Form ___ ____ structures within regions of the polypeptide chain.
•Two common regular secondary structures
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–
•Irregular structures are ___ ___ and ____

Answer 8

•Form recurring localized structures within regions of the polypeptide chain.
•
•Two common regular secondary structures
–a-Helix
–b-sheet
–
•Irregular structures are loops, turns and coils.
•

Question 9

The a-Helix

• a-Helices are ___ handed structures.
• Peptide backbone of the helix is formed by ____ between each ______ and the _______atom ___ residues apart. (Occurs within the ____)
• Found in____,___ ____and ___ ____proteins.
• _____is absent in helical region, since it doesn’t _____ and ____

Answer 9

• a-Helices are right handed structures.
• Peptide backbone of the helix is formed by H-bonds between each carbonyl oxygen atom and the amide hydrogen atom 4 residues apart. (Occurs within the same strand)
• Found in globular, membrane spanning and DNA binding proteins.
• Proline is absent in helical region, since it doesn’t posses an amide nitrogen for H-bonding and the appropriate bond angles to fit within the helix.

Question 10

b-Sheet
•b-sheets are composed of ____/____
•The strands/chains are held together by _______. Optimal H-bonding occurs when the strands are _____(____)
•These strands can run in ___ or ____
•Anti-parallel arrangement is composed of the ____ that _____
•In anti-parallel strands the atoms involved in H-bonding are_______

While the atoms in involved in H-bonding in the parallel arrangement are _____, one amino acid is H-bonded to ____ amino acids in the opposite strand.

Answer 10

•b-sheets are composed of strands/chains.
•The strands/chains are held together by H-bonding. Optimal H-bonding occurs when the strands are pleated (bent)
•These strands can run in the opposite (anti parallel) or in the same direction (parallel).
•Anti-parallel arrangement is composed of the same strand that folds back on it’s self.
•In anti-parallel strands the atoms involved in H-bonding are directly opposite to each other. While the atoms in involved in H-bonding in the parallel arrangement are skewed, one amino acid is H-bonded to two amino acids in the opposite strand.
•
•
•
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Question 11

Irregular Secondary Structures of Proteins

• b-turns consist of _______that connect ____ in ______ arrangement.
• In turns, the _____ residue is always ____, while the _____ is ____
• Loops are generally _____ residues long.
• Both loops and turns are found on the _____ of proteins which are involved in _____

Answer 11

• b-turns consist of 4 successive residues that connect b-strands in anti-parallel arrangement.
• In turns, the second residue is always proline, while the 3rd is glycine.
• Loops are generally 6 - 16 residues long.
• Both loops and turns are found on the surfaces of proteins which are involved in interaction with other molecules.

Question 12

Motifs

•Within a polypeptide chain there are _________________________known as structural motifs.

Similar structural motifs can be seen in ______.

•Example: _______ motif found in ___________. The ________structural motif is found within a domain of the enzyme _________

Answer 12

•Within a polypeptide chain there are arrangement of secondary structure that form a pattern known as structural motifs.

Similar structural motifs can be seen in different proteins.•

Example: Helix turn helix motif found in DNA binding proteins.

The ba ba b structural motif is found within a domain of the enzyme lactate dehydrogenase.

Question 13

Tertiary Structure of a Protein

• ______ of the secondary structures.
• Specifies the locations of______ in a protein.
• The structural elements fold into a___________
• The structures are not ___. They are_____ and _____. (bonds continually______)
• Types of bonding that holds the 3-D structure are ______, _____, ______ and _______.

Answer 13

• Folding of the secondary structures.
• Specifies the locations of each atom in a protein.
• The structural elements fold into a three dimensional structure.
• The structures are not rigid. They are dynamic and flexible.
• Types of bonding that holds the 3-D structure are Ionic interactions, H-bonds, disulfide bonds and hydrophobic interactions.

Dynamic bc bonds continually make and break

Question 14

Interactions that Stabilize Protein Structures

• Ionic interaction occurs between amino acid whose side chains are___
• Disulfide bridges are formed between______within and between polypeptide chains.

_____ of the sulfhydryl groups of the free cysteine residues results in the formation covalent disulfide bridges.

Answer 14

•Ionic interaction occurs between amino acid whose side chains are charged (Acidic and Basic amino acids).
•
•Disulfide bridges are formed between two cysteine residues within and between polypeptide chains. Oxidation of the sulfhydryl groups of the free cysteine residues results in the formation covalent disulfide bridges.

Question 15

Insulin Contains Two Polypeptide Chains Connected By Disulfide Bonds
Chain _ and _ are connected by __ disulfide bridge (_____), _____ disulfide bridge exist within chain __ (____)

Answer 15

Chain A and B are connected by two disulfide bridge (intermolecular), a single disulfide bridge exist within chain A (intramolecular)

Question 16

Interactions that Stabilize Protein Structures

•H-bonding occurs between amino acids with ____ and _____ side groups.
H-bonding also occurs between unpaired polar ___and ___ groups of the main chain to exist in hydrophobic environments. (___&____)

• Hydrophobic interactions. Strong tendency for hydrophobic amino acid residues to _____ _____resulting in the ____ of _____. An increase in ____ gives rise to a ____ _____ overall structure.
• Van der Waals interaction. The ___ structure of a protein is further stabilized by these forces between the____ _____ ______side chains.

•

Answer 16

•H-bonding occurs between amino acids with polar and charged side groups. H-bonding also occurs between unpaired polar NH and CO groups of the main chain to exist in hydrophobic environments. (a-helices, b-sheets)
•
•Hydrophobic interactions. Strong tendency for hydrophobic amino acid residues to cluster together resulting in the expulsion of water. An increase in entropy gives rise to a energetically stable overall structure.

•Van der Waals interaction. The compact structure of a protein is further stabilized by these forces between the tightly packed hydrocarbon side chains.

•

Question 17

Tertiary Structure of a Protein

• In cytosolic globular proteins the polar amino acids are found on the_____ of the protein while the non-polar or hydrophobic are found within the _____ (Ex_________)
• In Transmembrane proteins the non-polar amino acids are on the ____while the polar residues face the _____ of the protein lining ___ ____ channels. (Ex_______; ______)

Answer 17

• In cytosolic globular proteins the polar amino acids are found on the surface of the protein while the non-polar or hydrophobic are found within the core of the protein. (Myoglobin)
• In Transmembrane proteins the non-polar amino acids are on the surface while the polar residues face the interior of the protein lining water filled channels. (Bacterial porins; b-barrels)

Question 18

Tertiary Structure with a-Helix

Ex)
FCN

Answer 18

Example – Iron binding protein Ferritin is built entirely of a-helixes. Helps store iron.

Question 19

a-Helices Coiled-Coils

____or ____ a-helices______ ____ ____ _____to form a stable structure.

Found in _____, ______ and other structural proteins

Answer 19

Two or more a-helices entwined with each other to form a stable structure. Found in Keratin, myosin and other structural proteins

Question 20

Collagen

• The _____-______ structure of collagen arises from ___ amino acids: ____ ____ _____
• Three polypeptide chains can _________________.
• Rigid _____-____ linkages disrupt the ___ of ___ ___ in an a helix, but ____ the rigid three-stranded collagen helix.
• Found in ____ ____ ____ and ____

Answer 20

• The triple-helical structure of collagen arises from three amino acids: glycine, proline, and hydroxyproline.
• Three polypeptide chains can twist together to form a three-stranded helix.
• Rigid peptidyl- proline linkages disrupt the packing of amino acids in an a helix, but stabilize the rigid three-stranded collagen helix.
• Found in tendons, bone, ligaments and dentin.

Question 21

Tertiary Structures With b-sheets

Ex)_______, ______ , ________

Answer 21

Example – (b-barrels) Fatty acids binding protein, bacterial porins

Question 22

Protein Domains

___ stretches of amino acids of a polypeptide chain that ____ ____ into distinct 3-D structures from the rest of the protein.
•Example:

Answer 22

• Continuous stretches of amino acids of a polypeptide chain that fold independently into distinct 3-D structures from the rest of the protein.
• Example: Cell surface protein CD4 consists of 4 identical proteins

Question 23

Quaternary Structure of Proteins

• Combination of ________ polypeptide chains (subunits) to forms a Quaternary structure.
• Many proteins function as ____ _____ _____
• The subunits are arranged in a ____ and ____ manner.
• A subunit is known as a _____.
• The subunits could be identical (homo) or different (hetero). [homopentameric protein – E. coli Formate transporter (____ _____ subunits) heteromeric protein – Heterotrimeric G proteins (3 subunits]
• Hemoglobin consists of __ polypeptide chains (_________)

Answer 23

• Combination of 2 or more polypeptide chains (subunits) to forms a Quaternary structure.
• Many proteins function as dimers, tetramers and oligomers.
• The subunits are arranged in a geometrical and spatial manner.
• A subunit is known as a protomer.
• The subunits could be identical (homo) or different (hetero). [homopentameric protein – E. coli Formate transporter (5 similar subunits) heteromeric protein – Heterotrimeric G proteins (3 subunits]
• Hemoglobin consists of 4 polypeptide chains a1, a2, b1 and b2.

Question 24

Protein Folding
•_____determines the final structure of a protein.
•The final stable 3D structure is known as the _____.
•Most proteins will _____ ____ _____into the native structure.
If proteins misfold they would ____, be________ by ______ _____ or be_____ by _____ (___ ____proteins).
•When a polypeptide chain ____ and ____ it will form several ____ ______ (____ energy) until it finds the confirmation with _____ native energy. These are ___ ____. (___ the process ___).
•In some instance proteins require _____ to ____ in ____ by ___________ Ex-___ _____

Answer 24

•Primary sequence determines the final structure of a protein.
•The final stable 3D structure is known as the native protein.
•Most proteins will fold by themselves into the native structure. If proteins misfold they would aggregate, be degraded by proteolytic enzymes or be refolded by chaperones (heat shock proteins).
•When a polypeptide chain folds and refolds it will form several metastable intermediates (high energy) until it finds the confirmation with lowest native energy. These are kinetic barriers. Slow the process down.
•In some instance proteins require heat shock proteins (chaperones) to assist in folding by overcoming these kinetic barriers. Ex- hsp60 and 70
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•
•
•

Question 25

Protein Denaturation •Factors such as ____ \_\_\_\_\_ and _____ of the _____ affect protein structure. •Temperature – When the temperature is increased the __ and __ \_\_\_\_ in bonds \_\_\_\_ •Changes in pH will disrupt ____ and \_\_\_\_ •\_\_\_\_ can disrupt hydrophobic interactions. •These factors will disrupt the \_\_\_\_resulting in loss of \_\_\_\_

Answer 25

* Factors such as pH, temperature and the nature of the solvent affect protein structure. * Temperature – When the temperature is increased the rotational and vibrational energies in bonds increases. * Changes in pH will disrupt ionic and H-bonds. * Solvents can disrupt hydrophobic interactions. * These factors will disrupt the 3-D structure resulting in loss of function.

Question 26

Problems in Protein Folding Contribute to Human Diseases •Prion proteins cause ____ \_\_\_\_ These proteins act as _____ for other ___ proteins to\_\_\_ and ___ \_\_\_\_ •Prion diseases are acquired by ____ (\_\_ \_\_\_disease) or through ____ or ____ \_\_\_\_ (Creutzfeldt Jakob Disease (CJD).

Answer 26

•Prion proteins cause neurodegenerative diseases. •These proteins act as templates for other prion proteins to misfold and resist degradation. • •Prion diseases are acquired by infections (mad cow disease) or through sporadic or heritable mutations (Creutzfeldt Jakob Disease (CJD).

Question 27

sPrions ## Footnote * There are ___ confirmations - the ____ form (PrPC) and the ____ forming form (PrPSC) * PrPC is predominantly consist of \_\_\_\_, while PrPSC is enriched with \_\_\_\_. * This conformation favors _____ of PrPSC into a ____ complex which is ____ to ____ \_\_\_\_\_ * PrPC under normal conditions does not\_\_\_\_ to \_\_\_\_because of the ___ \_\_\_\_ \_\_\_

Answer 27

•There are two confirmations - the non disease form (PrPC) and the disease forming form (PrPSC) •PrPC is predominantly consist of a-helices, while PrPSC is enriched with b-sheets. •This conformation favor aggregation of PrPSC into a multimeric complex which is resistant to proteolytic degradation. •PrPC under normal conditions does not convert to PrPSC because of the high activation energy. • •

Question 28

Summary •The overall protein structure is defined by the •\_\_\_\_ ____ and \_\_\_\_are the main forces that stabilize the 3-D structure of a protein. •The 3-D native structure of a protein has the ____ energy (most stable structure). •The folding of proteins may or may not be assisted by _____ (heat shock proteins). •

Answer 28

•The overall protein structure is defined by the primary, secondary, tertiary and quaternary structures. •H-bonding, hydrophobic interactions and van der Waal’s interactions are the main forces that stabilize the 3-D structure of a protein. •The 3-D native structure of a protein has the lowest energy (most stable structure). •The folding of proteins may or may not be assisted by chaperones (heat shock proteins). •