CHAPTER 3 : PROTEINS

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PROTEINS ARE BUILT FROM REPERTOIRE OF AMINO ACIDS

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3.1 LEVELS OF PROTEIN STRUCTURE-Individual protein molecules can be described by…

“ up to four levels of structure”

: primary structure : : secondary structure, tertiary structure and quatenary structure :

linear sequence of amino acid The 3D structures residues in protein Forces responsible for maintaning OR

stabilizing these 3 levels are primarily “noncovalent”

PROTEOMICS the study of large sets of proteins

PROTEINS (structurally) Globular (e.g. enzymes)

Fibrous/ structural proteins (e.g. α-keratin)3

FOUR LEVELS OF PROTEIN STRUCTURE

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PROTEIN MODELS

STICK

3D RIBBONSPACE FILLING

CARTOON

FLAT RIBBON

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1) PRIMARY STRUCTURE

-Defined as the linear arrangement/ specific sequence of amino acids in a polypeptide chain linked through covalent peptide bonds. -In order to function properly, peptides and proteins must have the correct sequence of amino acids- Sometimes called "covalent structure of proteins” = all of the covalent bonding

within proteins defines the primary structure.

-In contrast, the higher orders of proteins structure (i.e. secondary, tertiary and quaternary) involve mainly non-covalent interactions.

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- In the protein hormone insulin, 51 amino acids are found

- Using 51 amino acids there are 1.55 x 1066 different possible sequences

- Many other proteins contain many more amino acids then insulin, but only the correct precise sequence is produced by the body

-The procedure used to synthesize the correct sequence of amino acids in proteins is guided by the genetics of DNA and RNA.

e.g. INSULIN

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2) SECONDARY STRUCTURE

1. Long chains of amino acids will commonly fold or curl into a regular repeating structure

2. Repeating conformations of peptide chains which are stabilised by hydrogen bonds between H of amino groups and O of carbonyl group of the peptide backbone

DEFINITION

This structure will add new properties to a protein like strength, flexibility and etc…

Protein conformation = local conformation of the backbone

= 3D arrangement of various atoms of the molecule

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Carbonyl: carbonyl functional group which is a divalent group consisting of a carbon atom with a double-bond to oxygen

Amide: a functional group containing a carbonyl group linked to a nitrogen atom.

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a) α helices

b) β structure (β strands and β sheets)

c) β turn

common secondary structure

a) Resembles a ribbon wrapped around a tube

(similar to a circular staircase)

b) Hydrogen bonds are found parallel to the helix axis

c) Very stable but flexible

(therefore it is often seen in parts of a protein that need to bend or move)

a) Two or more ribbons of amino acids are involved b) Hydrogen bonds are formed between the two (or more) polypeptide

strands

c) These line up to form a pleated like structure (similar to folds in fabric)

d) Tends to be rigid but less flexible than α helices.

• A structure in which the polypeptide backbone folds back on itself

• Often responsible for;-

-sharp bends and twists (in α helices) - hair-pins (in β sheets)

c) Useful for connecting helices and sheets

parallel

anti-parallel

right-handed screw

left-handed screw

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THE α HELIX-A spiral formed by coiling of the polypeptides chain around the fibre axis

-α helix present in more complex globular proteins

-All carbonyl group (in α helix) point toward C-terminus

-Average content of α helix in the proteins = 26%

-Bottom : N-terminus

-Top : C-terminus

-The α helices found in proteins are almost right-handed

“right handed α helix”

the backbone turns in a clockwise direction (when viewed along the axis from its N-terminus)

If you imagine that the right handed helix is spiral staircase, you will be turning to the right as you walk down the staircase

-blue ribbon indicates the shape of the polypeptide backbone

- All the side chains (R groups) project outward from the helix axis15

HYDROGEN BOND IN A HELIX

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HYDROGEN BOND IN A HELIX

H bond

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β SHEETS

-Hydrogen bonding occurs between neighbouring polypeptide chains rather than within one as in α helix

-Fibrous and insoluble in aqueous solvents

-This class includes ii) β SHEETSi) β STRANDS

β STRANDS -Portions of the polypeptide chain that are most fully extended

-Proteins rarely contain isolated β strands because the structure by itself is not significantly more stable than other conformations

β SHEETS

When multiple β strands are arranged side-by-side

-Are stabilised by H bonds between carbonyl oxygen and amide hydrogens on adjacent β strands

-β sheets sometimes called as β pleated sheet

-A typical β sheet contains from 2 to as many as 15 individual β strands 18

β SHEETS

b) anti-parallela) parallel

The hydrogen bonded chains extended in the

same directions

Neighbouring hydrogen bonded polypeptide chains run in

opposite directions

-running in opposite N- to C- terminal directions

-the H bonds essentially perpendicular to the β strands

-the space between H-bonded pairs alternatively wide and narrow

-Running in the same N- to C- terminal direction

-the H bonds not perpendicular to the β strands

-The H bonds are evenly spaced but slanted

-Are less stable than anti-parallel sheets 19

TURNS and LOOPS

In α helix or β strand Consecutive residues have a similar conformation that is repeated throughout the structure

turns and loops

-Regions that found in proteins which contain stretches of non-repeating 3D structure

-These regions can cause directional changes in the polypeptide backbone

-Connect α helices and β strands

-Allow the polypeptide chain to fold back on itself, producing 3-dimensional shape seen in native structure

BUT

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TURNSLOOPS

Usually found on the surfaces of proteins

(often contains hydrophilic residues)

-Loops containing only a few (up to five) residues will be referred as turns if they cause an abrupt change in the direction of a polypeptide chain

-β turns (or reverse turns);-

a) the most common types of tight turns b) usually connect different antiparallel β strands c) common types of β turns are;- “TYPE I” and “TYPE II”

(both types produce an rapid/ abrupt change in the direction of the polypeptide chain)

-All turns have internal hydrogen bonds that stabilize the structure

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LOOPS

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β TURNS

Occur more than twice as frequently as type II Always have Gly as the third residue

“Note that the hydrogen bonds

between

the peptide groups of the 1st and 4th residues of the bends”

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During and after protein synthesis, a protein folds into α helices and β sheets

These areas of secondary structure bind together and fold on each other in specific ways

Once the process of protein synthesis is completed, the protein takes its final shape. This stable form of the protein is known as the mature form, also known as the tertiary structure

3.3 TERTIARY STRUCTURE OF PROTEINS

by DEFINITION….

1) The shape of fully folded polypeptide chains

2) Results from the folding of its secondary structural elements

(which may already posses some regions of α helix and β structure)

into a closely packed 3D structure24

TERTIARY STRUCTURE

…important feature of tertiary structure…Amino acid residues that are far apart in the primary structure are brought

together, permitting interactions among their side chains

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2 types of tertiary structures:

1)Supersecondary structures (MOTIFS)2)Domains

TERTIARY STRUCTURE

-Most native proteins occurs at this state.

26Native protein = the naturally occurring form of protein

TERTIARY STRUCTURE STABILIZED BY..

so that distant regions of the chain are brought closer

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TERTIARY STRUCTURE

-Disulfide bridge though covalent, are also element of tertiary structure (they are not part of the primary structure since they form only after the protein folds)

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EXAMPLES OF TERTIARY STRUCTURE

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COMMON TYPES OF TERTIARY STRUCTURES

(a) SUPERSECONDARY STRUCTURES (a.k.a motifs)

-Are recognizable combinations of α helices + β strands + loops (that appear in a number of different proteins)

-Sometimes motifs are associated with a particular function (although structurally similar motifs may have different functions in different proteins)

-One of the simplest motifs

-Occurs in a number of calcium-binding proteins

(since Glutamate and Aspartate residues in the loop of calcium-binding proteins form part of the calcium-binding site)

-Also called as helix-turn-helix in certain DNA-binding proteins

(since the residues that connect the helices form a reverse turn. The α helices residues in DNA-binding proteins bind DNA)

…common motifs…

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-Consists of two amphipathic α helices that interact through their hydrophobic edges

-- e.g. ;- leucine zipper

-The individual α helices have opposite orientations

-Parallel in the coiled-coil motif

-Consists of two parallel β strands linked to an intervening

α helix by 2 loops

- The helix connects the C-terminal end of one β strand to the N-terminal end of the next

-Often runs parallel to the two strands 31

- Consists of 2 adjacent antiparallel β strands connected by a β turn

-Is an antiparallel β sheet composed of sequential β strands connected by loops or turns

-May contain one or more hairpins but, more typically the strands are joined by larger loops

- This is a β sheet motif linking four antiparallel β strands such that;-

strands 3 and 4 (form the outer edges of the sheet) strand 1 and 2 (are in the middle of the sheet)

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-Is formed when β strands or sheets stack on the top of one another

-Figure shows an example of a β sandwich where the β strands are connected by short loops and turns..

BUT

β sandwich can also be formed by the interaction of 2 β sheets in different regions of the polypeptide chain

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(b) DOMAINS

-Are compact units which are discrete and independently folded

-May consist of combinations of motifs

-Are usually connected by loops,

BUT

they are also bound to each other THROUGH weak interactions

(formed by the amino acid side chains on the surface of each domain)

-Some domain structures occur in many different proteins while others are unique

COMMON TYPES OF TERTIARY STRUCTURES

In general, proteins can be grouped into families according to…

“ similarities in domain structure and amino acid sequence”34

CLASSIFICATION OF DOMAINS

- Commonly used classification scheme groups are;-

α category domains consist almost entirely of α helices and loops

β category domains consist only β sheets and nonrepetitive structures that link β strands

α/β category domains have supersecondary structures such as the βαβ motif and others (in which regions of α helix and β strand alternate in the polypeptide chain)

α+β category domainsconsist of local clusters of α helices and β sheet (where each type of secondary structure arises from separate contiguous regions in the polypeptide chain)

- Within each of the 4 main structural categories, protein domains can be further classified by the presence of characteristic folds;-

folds Is a combination of secondary structures that form the core of a domain

“ some domain have easily recognizable folds such as the β meander. However, other folds are more complex”

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COMMON DOMAIN FOLDS

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3.4 QUATERNARY STRUCTURE

-Many protein exhibit an additional level of organization called as quaternary structure

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by DEFINITION…

(1) Quaternary structure refers to the organization and arrangement of subunits

… IN …

a protein with multiple subunits

multisubunit protein = an oligomer (proteins with only one polypeptide chain are monomers)

QUATERNARY STRUCTURES

(2) Quartenary structure is the stable association …OF... multiple polypeptide chains resulting in an active unit

Not all proteins exhibit quartenary structure

Usually, each polypeptide within a multisubunit protein folds more-or-less independently into a stable tertiary structure

the folded subunits then associate with each other to form the final structure

(quaternary structure) 38

SUBUNITS

-each subunit = a separate polypeptide chain

-the subunits within an oligomeric protein always have a defined stoichiometry

-the arrangement of the subunits give rise to a stable structure

-the subunits of a multisubunit protein may be…

identical different

Dimers and tetramers predominate Each type often has a different function

common shorthand method for describing oligomeric proteins

Uses ;-a) Greek Letters to identify types of subunits

b) Subscript numerals to indicate number of subunits

Example ;-α2βγ protein contains 2 subunits designated α

EACH 1 of subunits designated β and γ39

SUBUNITS

-Subunits are held together by many weak, noncovalent interactions

electrostatic forceshydrophobic interactions

-principle forces involved -contribute to the

proper alignment of the subunits

- The subunits of an oligomeric protein can be often separated in the laboratory because…intersubunit forces are usually rather weak…

ionic bondingHydrogen bonding

Van der Walls interaction

involved in the interactions between subunits

in rare instancesbetween cysteine residues in different polypeptide chainsdisulfide bonds

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EXAMPLE OF QUATERNARY STRUCTURE

-This protein has 2 identical subunits with α/β barrel folds

-The identical subunits associate through weak interactions between the side chains found mainly in loop regions 41

EXAMPLE OF QUATERNARY STRUCTURE

-This protein has identical all-β subunits that bind symmetrically

-The identical subunits associate through weak interactions between the side chains found mainly in loop regions 42

Determination of the subunit composition of an oligomeric protein

-essential step in the physical description of a protein

a) Molecular weight of native oligomer estimated by gel-filtration chromatography

b) Molecular weight of each chain SDS-polyacrylamide gel electrophoresis

SUBUNITS

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3.5 PROTEIN DENATURATION AND RENATURATION

DENATURATION

Disruption of the protein native conformation, commonly caused by

HEATING (but only with small range of temperature)

Can cause loss of biological activity since the protein will malfunction

Amount of energy needed for the process is often small

(perhaps equivalent to the energy needed for the disruption of 3 @ 4 hydrogen bonds)

as a result

Some of the denatured proteins ;-

-May unfold completely to form a random coil

-Small denatured proteins can spontaneously renature, or refold

Most of the denatured proteins ;-

Retain considerable internal structure

Is a cooperative process the destabilization (of just a few weak interactions) leads to

almost complete loss (of native conformation)44

How DENATURATION can occur ??

Thermal

Modest increase in temperature will result in unfolding and loss of secondary and tertiary structure

Requires a reducing agent that disrupts disulfide bridge, which will finally allowing protein to unfold

1 Most proteins are stable at temperatures up to 50oC to 60oC

Chaotropic Agents2e.g. urea and guanidinium salts

By allowing molecules to solvate nonpolar groups in the interior of proteins

Detergents3 The water molecules disrupt the hydrophobic interactions that normally stabilize the native conformations

e.g. hydrophobic tails of detergents sodium dodecyl sulfate

Denature proteins by penetrating the protein interior and disrupting hydrophobic interactions

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PROTEIN DENATURATION

Thermal denaturation of horse apomyoglobin and ribonuclease A

(the midpoint of T range over which denaturation occurs = Tm) 46

Most proteins have a characteristics “melting” T (Tm) that corresponds to the T at the midpoint of the transition between the native and denature forms

Tm depends on pH and the ionic strength of the solution

RENATURATION

For this case, renaturation of Ribonuclease A can occur if :-

a) Urea is removed

b) Small amount of 2-mercaptoethanol is added

Occur at Tm ;-

Regain its native conformation

Regain correct set of disulfide

bonds

Regain full enzymatic activity

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3.6 COLLAGEN

3.7 STRUCTURES MYOGLOBIN ND HEMOGLOBIN

3.8 ANTIBODIES AND BIND AND SPECIFIC ANTIGENS

-2 groups (a group of 5) for each sub topic

-Reference : Text Book Only

-Presentation Date : Week 10

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