Summary of last lecture (recorded)

Amino acids are joined by peptide bonds Differences in protein function result from differences in amino acid composition and sequence. Secondary structure: alpha-helix and betasheets Wide variety of 3D structures formed by folding of various sections of secondary structure. Proteins consist of Domains

Domains

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Conus victoriae Courtesy of Bruce Livett & David Paul Tying the ends of a protein together makes the molecule resistant to exopeptidase enzymes that chomp away at peptide termini. But Craik says the cyclization also enhances hydrogen bonding across the entire molecule, making it resistant to the endopeptidases that attack a proteins interior amino acids. He says its sort of like a zipper: A zipper can be regarded as a series of hydrogen bonds all interlocking together, and when you zip it all up, youve got a beautiful set of coordinated hydrogen bonds. But youve still got two ends, and when you pull apart those two ends of the zipper, then the first hydrogen bond goes, then the next, and then the next.

http://pubs.acs.org/cen/science/88/8830sci2.html

The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain
Clark R et al, Angewandte Chemie, 2010.

All of the following are correct about a peptide bond except:

1. 2. It exhibits partial doublebond character It is more stable in the cis configuration than in the trans configuration It has restricted rotation around the carbonyl carbon to nitrogen bond It is planar

3.

4.

-helices are stabilised by:

1. Adjacent negatively charged amino acids 2. Glycine residues 3. Large bulky amino acids close together 4. Hydrogen bonding

In the -helix the hydrogen bonds:

1. 2. 3. are roughly parallel to the axis of the helix. are roughly perpendicular to the axis of the helix. occur mainly between electronegative atoms of the R groups. occur only between some of the amino acids of the helix. occur only near the amino and carboxyl termini of the helix.

4. 5.

Thr and/or Leu residues tend to disrupt an a-helix when they occur next to each other in a protein because:
1. 2. 3. 4. 5. an amino acids like Thr is highly hydrophobic. covalent interactions may occur between the Thr side chains. electrostatic repulsion occurs between the Thr side chains. steric hindrance occurs between the bulky Thr side chains. the R group of Thr can form a hydrogen bond.

-sheets are held in a side by side arrangement mainly by:

1. disulfide bonds 2. H-bonding 3. interactions between charged side chains. 4. lysine cross-links 5. none of the above

Proline residues are usually present in:

1. 2. 3. 4.

-sheets -sheets -turns -helices

Protein Structure & Function

Tertiary structure 2 major groups of proteins Fibrous with polypeptide chains in long chains or sheets Globular with polypeptides folded into more or less spherical shape
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Fibrous proteins
1. Keratins (-helical proteins) 2. Fibroin (-conformation) 3. Collagen (triple helix) 4. Elastin

These proteins give strength and flexibility to the structures in which they occur. Fundamental structure is simple repeating element of secondary structure.
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-keratins
Major protein components of hair and fingernails as well as comprising a major part of animal skin. -keratins are part of a broader family of proteins called intermediate filament proteins that play important structural roles in the cell cytoskeleton. Consist entirely of -helices. The -helices are cross-linked by disulfide bonds.

http://www.immediart.com/catalog/images/bigger_images/SPL_6 _P780110-Fibroblast_cells_showing_cytoskeleton.jpg

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-keratins
Hair consists of fibrils, which consist of even smaller fibres twisted around each other. Each fibre consists of 2 -helical polypeptides oriented in parallel) coiled around each other to form a super-twisted coiled coil. The super-twisting amplifies the strength of the overall structure.

www.imbjena.de/.../proteins_classification.html
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-keratins
The helical path of the super-twist is left-handed, the opposite to the individual -helices. The surfaces where the two -helices touch are made up of hydrophobic residues with their R groups meshed together in a regular interlocking pattern. This permits close packing of the polypeptide chains within the left-handed supertwist. -keratin is rich in the hydrophobic residues Ala, Val, Leu, Ile, Met and Phe.

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-keratins

About 4 protofibrils 32 strands of -keratin combine to form an intermediate filament. A hair is an array of many -keratin filaments.

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-keratins

The intertwining of the two -helical polypeptides is an example of quaternary structure. The quaternary structure of -keratin can be quite complex with many coiled coils assembled into large supramolecular complexes (eg: filament of hair). In -keratin the cross-links stabilizing the quaternary structure are disulfide bonds.

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-keratins cross-linking

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Permanent waving of hair

Bend hair around an appropriate shape

Moist heat breaks hydrogen bonds

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Oxidizing agent makes new disulfide bonds

Properties of -keratins
The -helical structure of -keratins is extendable but this depends on the degree of cross-linking. Therefore the tougher the -keratin, the higher degree of disulfide cross-linking (differences between hair and fingernails) Hardest and toughest is rhinoceros horn -keratin, which has 18% of the residues as cysteines involved in disulfide bonds. -keratins are rich in hydrophobic residues which extend outward from the -helices, and thus are completely insoluble in water.
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-conformation
-keratins are stringy, insoluble proteins.
Beta-keratins form the hard corneous material of reptilian scales Toni et al, J. Proteome Res., 2007, 6 (9), pp 33773392

Autumn and Peattie (2002) Integrative Comp. Biol. 42, 1081

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-conformation
Fibroin, the component of silk and spider webs. Consist of mostly -pleated sheet. Rich in small amino acid residues such as glycine and alanine permitting close packing of -sheets and an interlocking arrangement of R groups. The overall structure is stabilized by extensive hydrogen bonding between all peptide linkages in the peptides of each -sheet and by optimization of van der Waals interactions between sheets.

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Silk fibroin
Composition of silk fibroin is 36% gly, 24% ala, 12 % ser (ratio 3:2:1) consisting of repeats of (gly-ser-gly-alagly-ala)n along the lengths of the polypeptides. The -sheets are antiparallel containing small amino acids packed tightly together and stabilized through extensive hydrogen bonding to form strong inextensible strands.

hmaloy.wikispaces.com
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Silk fibroin

http://en.wikipedia.org/wiki/Spider_silk

Courtesy of University of Cambridge, UK Department of Materials Science

Silk is remarkably strong, with a tensile strength similar to high grade steel.

Silk does not stretch because it is already highly extended. However, the structure is flexible because it is held together by numerous weak interactions rather than the by covalent bonds such as disulfide bonds.
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Silk fibroin

The fibers of silk and spider web are made up of the protein fibroin, which consists of layers of anti-parallel sheets rich in Ala and Gly residues.
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Silk fibroin

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Collagen
Is the most abundant protein in most vertebrates providing the strength to connective tissues such as tendons, cartilage, the organic matrix of bone and is an important constituent of the skin. The collagen helix is unique and quite distinct from an -helix. It is a left-handed helix consisting of 3 residues per turn Collagen is also a coiled coil with distinct tertiary and quaternary structures. Three chains are twisted around each other and this superhelical twist is right-handed.
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Collagen chain
Left-handed helix with 3 residues per turn. The sequence has a glycine residue in every 3rd position and a high percentage of proline and hydroxyproline. Every 3rd residue of the helix lies in the centre of the triple helix. Due to size constraints this must be a glycine. The amino acid sequence of collagen is generally a repeat of gly-X-Y where X is often pro and Y is often hydroxyproline. Pro and hydroxyproline permit the sharp twisting of the helix.
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Collagen triple helix (Tropocollagen)

The tight wrapping of the alpha chains in the collagen triple helix provides tensile strength greater than that of steel wire of equal cross section.

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Collagen triple helix (Tropocollagen)

Tropocollagen is the basic unit of the collagen structure. Hydrogen bonding between the 3 chains stabilises the tropocollagen triple helix. Hydroxypro provide more avenues for hydrogen bonding. Tropocollagen molecules pack side by side in an overlapping fashion and are cross-linked to each other via lysine residues to form a collagen fibre. This gives the structure incredible strength.

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Collagen triple helix (Tropocollagen)

Fig. 4-11. Glycine is shown in red. Because of its small size is required at the tight junction where the three chains are in contact.

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Collagen fibrils
The tight wrapping of the of the chains in the collagen triple helix provides tensile strength greater then that of steel wire of equal cross section. Collagen fibrils are supramolecular assemblies consisting of the triple-helical collagen molecules associated to provide different degrees of strength. Tropocollagen molecules pack side by side in an overlapping fashion and are cross-linked to each other via lysine, 5-hydroxylysine and histidine residues that are present at a few of the X and Y positions in collagen. The rigid and brittle nature of aging is due to an accumulation of covalent cross-links.
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Collagen fibrils

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Collagen fibrils

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Collagen defects
Some human genetic defects illustrate the close relationship between amino acid sequence and 3-dimensional structure. Osteogenesis imperfecta (abnormal bone formation in babies) and Ehlers-Danlos syndrome (loose joints) are both conditions that can be lethal and both result from the substitution of an amino acid with a larger R group (such as Cys or Ser or Arg) for a single Gly residue in each chain These single substitutions have catastrophic effect on collagen function because they disrupt the Gly-X-Y repeat that gives collagen its unique structure.

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Ehlers-Danlos syndrome (EDS)

Family of diseases caused by abnormalities in the synthesis and metabolism of collagen

decrease in the tensile strength and integrity of the skin, joints, and other connective tissues.
"The India Rubber Man, The Elastic Lady," and "The Human Pretzel."

Disease associated with abnormal fibrous proteins

Epidermolysis Bullosa

Keratins and Collagens (type VII) and others Keratins

Epidermolytic Hyperkeratosis

ebworld.faithweb.com/clinicalpics_j.htm

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Elastin
Found in tissues such as ligaments arterial blood vessels which require highly elastic fibres. The polypeptide of elastin is rich in glycine, alanine and valine, and is very flexible and easily extended. Conformation is random coil which has little or no secondary structure. The polypeptide also contains frequent lysine residues which cross-link polypeptide chains. The cross-links prevent the elastin fibres from extending indefinitely, allowing them to snap back when the tension is relieved.
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Elastin

contains mostly Gly, Ala and Val with some Lys and Pro
www.imb-jena.de/.../proteins_classification.html
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Ionization of Peptides

The acid-base behaviour of a peptide can be predicted from its free a-amino and a-carboxyl groups as well as the nature and number of its ionizable R groups. Like free amino acids, peptides have characteristic titration curves and isoelectric points pI pH at which they carry no nett charge, and do not move in an electric field).
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When the [H+] changes from 10-3 M to 10-5 M, the pH ..

1. Increases by 2 units 2. Decreases by 2 units 3. Stays the same 4. Increases 100 x 5. Decreases 100 x

To a container holding 999mL water, 1.0 mL of 0.00001M HCl is added, what is the final pH?

1. 2. 3. 4.

5 7 9 3

To a container holding 999mL water, 1.0 mL of 0.001M NaOH is added, what is the final pH?

1. 2. 3. 4. 5.

6 7 8 10 11

At pH 7.0 alanine has a net charge of ..

1. +1 2. 0 3. -1

Ionisation of amino acids

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Ionisation of amino acids

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An amino acid that has a charged side group at pH 13 is:

1. 2. 3. 4. 5.

leucine Lysine Glutamine Aspartic acid Glycine

What percentage of a weak acid is ionized at a pH value equal to its pKa? 1. 2. 3. 4. 5. 10% 25% 50% 75% 90%

The pKa of the histidine side chain is 6.0. What is the ratio of the acid form to the conjugate base form at a pH of 8?

...

1. 2. 3. 4. 5.

0.01 0.1 1 10 100

Net charge of a peptide

Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ?

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Net charge of a peptide

Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ? NH3+-A-E-I-L-K-V-G-COOH 1. Remember that every peptide has an N-terminus and a C-terminus.

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Net charge of a peptide

Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ? NH3+-A-E-I-L-K-V-G-COOH 2. Identify any amino acids that have a side chain that can be ionised, such as side chains with positively and negatively groups. In the case of the above peptide this is Lysine (K) and Glutamic acid (E).

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Net charge of a peptide

Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ? pKa NH3+-A-E-I-L-K-V-G-COOH 9.7 4.3 10.5 2.3

3. Consider the pKa of each of the ionisable groups. pH > pKa the proton tends to be off pH < pKa the proton tends to be on. pH = pKa half of the molecules are protonated and half are deprotonated.

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Net charge of a peptide

Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ? pKa pH 3.0 NH3+-A-E-I-L-K-V-G-COOH 9.7 4.3 10.5 2.3 +1 0 +1 -1 Net charge +1

pH > pKa the proton tends to be off. pH < pKa the proton tends to be on. pH = pKa half of the molecules are protonated and half are deprotonated.

1002BPS STRUCTURAL BIOCHEMISTRY MODULE 2

Net charge of a peptide

Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ? pKa pH 3.0 pH 8.0 pH 12.0 NH3+-A-E-I-L-K-V-G-COOH 9.7 4.3 10.5 2.3 +1 +1 0 0 -1 -1 +1 +1 0 -1 -1 -1 Net charge +1 0 -2

pH > pKa the proton tends to be off. pH < pKa the proton tends to be on. pH = pKa half of the molecules are protonated and half are deprotonated.
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What is the net charge of the peptide Val-Gly-Asn-Ala at neutral pH (pH 7.0)?

1. 2. 3. 4.

+1 0 -1 Unable to determine the charge 5. Havent got a clue

Consider the peptide ALRKRG. What is the charge on the peptide a pH 2?

1. 2. 3. 4. 5.

-4 -2 neutral +2 +4

Consider the peptide ALRKRG. What is the charge on the peptide a pH 2? 1. 2. 3. 4. 5. -4 -2 neutral +2 +4

Calculation of partial charge of amino acid residues

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Practice Problem What is the charge on the following peptide at pH 3, 7, 9 and 11? AHYNERL
Use the pKa values given on slide no. 52.

What is the approximate isoelectric point of this peptide?