CUBT105: INTRODUCTION TO

MOLECULAR BIOLOGY

PROTEIN STRUCTURE &

FUNCTION

TENDAI WALTER SANYIKA

CHINHOYI UNIVERSITY OF TECHNOLOGY, DEPARTMENT OF BIOTECHNOLOGY

ROOM 10, Block 11

waltersanyika@yahoo.com; wsanyika@cut.ac.zw

CUBT105- Protein Structure & Function – This lecture is describes and explains the

biochemistry of protein structure and function.

Specific Objectives:

1. To describe proteins as molecules of life made up of amino acid subunits.

2. To describe the protein structures:

– Primary, secondary, tertiary and quaternary structures.

Study Material

Textbook Reading:

1. Voet & Voet, Biochemistry.

2. Lubert Stryer, Biochemistry.

• OR any relevant biochemistry text book.

Proteins • Proteins are organic, biological molecules.

• Proteins are made of 20 amino acids linked by peptide bonds.

• Linked amino acids forms a polypeptide backbone. » Polypeptide has a polarity with repeating sequence (N to C

terminus).

» Amino acids repeatedly linked in the to C terminal via peptide bond.

• Proteins make up about 15% of the cell.

• Have many functions in the cell – Enzymes

– Structural

– Transport

– Motor

– Storage

– Signaling

– Receptors

– Gene regulation

– Special functions Models of protein structure

General Amino Acid Structure

Cα

H

R

COOH H2N

Side chain

Carboxyl group (C terminus);

acidic Amino group (N terminus);

basic

Hydrogen atom

General Amino Acid Structure At Isoelectric Point

(pH at which the net Charge = 0)

Cα

H

R

COO- +H3N

• There is an internal transfer of a hydrogen ion from the -

COOH group to the -NH2 group to leave an ion with both a

negative charge and a positive charge.

• This is called a zwitterion.

Amino Acid Acid-Base Properties

Amino Acid Distinctive Properties Depends on Properties of the Side Chain

(R- Group)

Hydrophilic Hydrophobic

Peptide Bond

Formation • The acid end of one

amino acid reacts with the amine end of another amino acid.

• Reaction releases a water molecule.

• Result is an amide bond, forming a dipeptide. (highlighted in blue color).

• Addition of another amino acid to the tripeptide, then tetrapeptide….etc.

• Continued elongation forms a polypeptide.

• Polypeptide folds into a functional protein.

The enzyme for peptide bond formation is

Peptidyl transferase, an aminoacyltransferase.

Peptide Bond Formation

Resonance Property of The Peptide

Bond • The peptide bond is rigid because of the resonance interaction

of the amide and carbonyl groups. – The ability of the amide nitrogen to delocalize its lone pair of electrons

onto the carbonyl oxygen.

• Peptide bond shows a rigid plane between the two flanking α-carbon atoms.

• There is no rotation around the C-N bond resulting in structural stability.

» Explained by the electronic resonance character of the O=C-N structure.

» Double bond character changes between the O-C and C-N bonds.

• These bonds do not appear as other similar bonds due to this resonance phenomenon.

» The C=O bond is actually longer than normal carbonyl bonds.

» The N–C bond of the peptide bond is shorter than the N–Cα bond.

• The carbonyl oxygen and amide hydrogen are in a trans (opposite sides) configuration, as opposed to a cis (same side) configuration.

» Configuration energetically more favorable – results in less steric interactions (congestion) of atoms.

Peptide Bond Resonance

Peptide Bond Resonance

Polypeptide

Backbone

Protein Shape Determined by Amino Acid Sequence

• The amino acid side chain or R group is not part of the

backbone or the peptide bond.

• The peptide bond allows for rotation around it – allows

flexibility. » The protein can therefore, fold and orient the R groups in

favorable positions

» Weak non-covalent interactions hold protein in functional

shape.

• Thus, proteins shape is determined by amino acid sequence.

• The final shape is called the conformation. » Has the lowest free energy possible.

• Denaturation is the process of unfolding the protein » Can be down with heat, pH or chemical compounds.

» Proteins can be denatured and renatured.

» Proteins can be cleaved by proteases and chemical

reagents.

Protein Conformation Framework • Bond rotation determines protein folding (3D

structure).

• Torsion angle (dihedral angle) τ – Measures orientation of four linked atoms in a molecule: A,

B, C, D

– τABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D

Backbone Torsion Angles

Preteases or Proteinases Enzymes That Cleave The Peptide Bond

• Belong to the class of enzymes known as hydrolases, which catalyse the reaction of hydrolysis of various bonds with the participation of a water molecule.

• They are specific.

• Standard Classification: – Proteases are currently classified into six broad groups:

– Serine proteases.

– Threonine proteases.

– Cysteine proteases.

– Aspartate proteases.

– Metalloproteases.

– Glutamic acid proteases.

Types of Proteins

• Globular Proteins. » Compact shape like a ball with irregular surfaces, eg.

enzymes.

• Fibrous Proteins – usually span a long distance in the cell.

» 3-D structure is usually long and rod shaped (usually have structural roles)

Examples of Fibrous Proteins • Actin filaments, intermediate filaments and

microtubules. » Integral components of the cytoskeleton system.

» Support cell structure and functions.

• Intermediate filaments of the cytoskeleton. » Structural scaffold inside the cell.

» Provide structural rigidity and tensile flexibility to cell.

» Eg. Keratin in hair, horns and nails.

• Cell extracellular matrix. » Bind cells together to make tissues

» Secreted from cells and assemble in long fibers

• Collagen - fiber with a glycine every third amino acid in the protein.

» Main component of connective tissues (approx. 25-35% of body protein).

• Elastin - unstructured fibers that gives tissue an elastic characteristic.

» Also component of connective tissue.

Collagen and Elastin

Fibrous Proteins

Cell extracellular matrix

Cytoskeleton system

Globular Proteins: Examples 1. Antibody Family • A family of proteins that can bind to almost any molecule.

• Antibodies (immunoglobulins) made in response to a foreign molecule called the antigen.

» Eg. bacteria, virus, pollen, etc.

• Specifically bind and inactivates the antigen or marks it for destruction.

• Important for functioning of the immune system – specific immune response.

2. Enzymes • Proteins that catalyze chemical reactions.

• Biological catalysts - they speed up the chemical reactions in living things.

» Lower the activation energy for a reaction.

» Dramatically increase the rate of the reaction.

• Examples: » Lysozyme, amylases, proteases, lipases, cellulases, alcohol

dehydrogenase, lactse, ………

Levels of Protein Organization • Primary structure.

» Amino acid sequence of the protein.

» Includes type of amino acid, their sequence and number of residues.

» Also includes disulfide bonds.

• Secondary structure. » Held by hydrogen bonds in the peptide chain backbone.

» -helix and -sheet structures formed in secondary structure.

»

• Tertiary structure. » Non-covalent interactions between the R groups within the

protein.

» Eg. Ionic and hydrophobic forces/ interactions.

• Quaternary structure. » Interaction between 2 folded polypeptide chains (proteins).

Biochemistry of Protein Structure

Primary

Secondary

Tertiary

Quaternary

Assembly

Folding

Packing

Interaction

S T

R U

C T

U R

E

P R

O C

E S

S

Protein Structure

Protein Primary Structure • Protein: chain of amino acids joined by peptide

bonds. » Linear.

» 1 dimensional.

» Ordered sequence of amino acids.

» sequence of amino acid polymer.

» Defined from N to C terminus.

» May contain disulfide bonds.

• By convention, polar; written from amino end to carboxyl end.

• A perfectly linear amino acid polymer is neither functional nor energetically favorable for folding.

• Type of amino acid and sequence important: – Especially the side chain (R-group).

Protein Primary Structure • The primary structure is the sequence of amino acids

that are: – Linked together.

– The linear structure is called a polypeptide.

http://www.mywiseowl.com/articles/Image:Protein-primary-structure.png

Disulfide Bonds • Side chain of cysteine contains highly reactive thiol (-

SH) group.

» Also known as the sulfhydryl group.

• Two thiol groups can form a disulfide bond.

• This contributes to the stability of the folded state by linking distant parts of the polypeptide chain. – Within the same primary structure.

Disulfide Bridge

• Reduction of thiol/ sufhydryl groups forms a disulfide bridge.

• It is a reduction reaction.

Disulfide Bridge

Can Link Distant Amino Acids

Protein Secondary Structure • Protein secondary structure characterized by two

features: – Alpha helices.

– Beta sheets.

• And also random coils – usually form the binding & active sites of proteins.

Source: http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/prot.htm#I

Secondary Structure • Non-linear.

• 2 dimensional.

• Localized to regions of an

amino acid chain.

• Formed and stabilized by

hydrogen bonding,

hydrophobic interactions,

electrostatic and van der

Waals interactions.

• Involves three atoms: • Donor electronegative atom. (D).

– (Nitrogen or Oxygen in proteins).

• Hydrogen bound to donor. (H).

• Acceptor electronegative atom (A) in close proximity.

• Polarization due to electron withdrawal from the

Hydrogen atom to side chain donor.

• Causes the donor to have a partial negative charge

and the H atom a partial positive charge.

• Proximity of the Acceptor A causes further charge

separation

• Result: – Closer approach of acceptor to Hydrogen (bonding).

Hydrogen Bonding

Hydrogen Bonds in Proteins

• H-bonds form between. – Atoms involved in the peptide bond.

– Peptide bond atoms and R groups.

– R groups.

Protein Folding

• 2 regular folding patterns

have been identified –

formed between the

bonds of the peptide

backbone

• -helix – protein turns like

a spiral – fibrous proteins

(hair, nails, horns)

• -sheet – protein folds

back on itself as in a

ribbon – globular protein

Hydrogen Bonding

And Secondary Structure

Alpha-helix Beta-sheet

Sheets • Core of many proteins is

the sheet.

• Form rigid structures

with the H-bond.

• Can be of 2 types: – Anti-parallel – run in an

opposite direction of its

neighbor (A).

– Parallel – run in the same

direction with longer looping

sections between them (B).

Helix

• Formed by a H-bond between every 4th peptide bond – C=O to N-H.

• Usually found in proteins that span a membrane.

• The helix can either coil to the right or the left.

• Can also coil around each other. – Coiled-coil shape – a

framework for structural proteins such as nails and skin.

Tertiary Structure • How the random coils, alpha

helices and beta sheets fold.

• Held in place by specific bonds: – Weak Hydrogen bonds

between amino acids that lie close to each other,

– Strong ionic bonds between R groups with positive and negative charges, and

– Disulfide bridges (strong covalent S-S bonds).

• Amino acids that were distant in the primary structure may now become very close to each other after the folding has taken place

• The subunit of a more complex protein has now been formed. It may be globular or fibrous. It now has its functional shape or conformation.

Source: io.uwinnipeg.ca/~simmons/ cm1503/proteins.htm

Tertiary

Structure • Non-linear but 3 dimensional.

• Folded protein.

• Globular but restricted to one

polypeptide polymer.

• Formed and stabilized by:

• Hydrogen bonding (including those from

secondary structure).

• Covalent bonding (disulfide bond from

primary structure).

• Hydrophobic packing toward core and

hydrophilic exposure to solvent.

• A globular amino acid polymer

folded and compacted is somewhat

functional (catalytic) and

energetically favorable for

interaction with other tertiary

structures.

• Specific overall shape of a protein.

• Cross links between R groups of amino acids in

chain:

- Disulfide: –S–S–

- Ionic: –COO– …………H3N+–

- H bonds: –C=O …………...HO–

- Hydrophobic: –CH3 …………... H3C–

Tertiary Structure Bonding

Protein Folding & Packing Folding:

• Proteins shape determined by the sequence of the amino acids. » Molecular chaperones are small proteins that help guide proteins to

fold into the correct conformation and assist with associating correct polypeptides together

• The final shape is called the conformation - has the lowest free energy.

Protein Packing:

• Usually occurs in the cytosol (~60% bulk water, ~40% water of hydration).

• Involves interaction between secondary structures & solvent.

• May be promoted by chaperones such as for membrane or membrane-bound proteins.

• Results in globule states » Overall entropy loss is small enough so enthalpy determines sign of E, which decreases (loss in entropy from packing counteracted by gain from desolvation and reorganization of water, i.e. hydrophobic effect)

• Packing yields tertiary structure.

Protein Folding

• The folding process is complicated, and happening

in the presence of high concentrations of other

proteins.

• Might need assistance of factors such as:

1. Metals.

2. Co-factors.

3. A different cellular location (periplasm?).

4. Disulfide-forming enzymes.

5. Chaperones.

• Proteins which help with hydrophobic sequences.

Trigger factor (Tf)

• binds 50S subunit;

• peptidyl-prolyl cis-trans isomerase

DnaJ/K

• binds nascent polypeptides;

• shield exposed hydrophobic

patches from folding unfavorably

GrpE

• binds polypeptides released from

DnaK/J and releases polypeptides

into folded form or shuttles to

GroEL/ES

GroEL/ES

• helps fold/refold proteins

already in compact state but are

not yet folded

Role of Molecular Chaperones In E. Coli

Protein Domain • Domains were first suggested to be independent folding protein

units (segments) by Richardson.

• Different criteria for assessing domain regions within a structure: » A compact globular structure

» Residues within a domain make more internal contacts than other regions

» Secondary Structure Elements are usually not shared with other regions

» An evolutionary unit

• In some (but not all) cases, each domain in a protein is encoded by a separate exon in the gene encoding that protein.

• A domain is a basic structural unit of a protein structure – distinct from those that make up the conformations.

» Part of protein that can fold into a stable structure independently.

» Usually part of the tertiary structure.

• Different domains can impart different functions to proteins

• Proteins can have one to many domains depending on protein size.

Quaternary Structure

Protein Quaternary Structure • This is association of the protein

subunits to form the final protein complex. – For example, the human hemoglobin

molecule is a tetramer made up of two alpha and two beta polypeptide chains (right).

Source: www.cem.msu.edu/~parrill/movies/neuram.GIF

• This is also when the protein

can be modified to associate

with non-protein groups. – Eg. Carbohydrates can be

added to form glycoproteins

(modification shown in green).

Source: www.ibri.org/Books/ Pun_Evolution/Chapter2/2.6.htm

Tertiary or Multi-Subunit Proteins Quaternary Structure

Prosthetic Group

• Occasionally the sequence of the protein is not enough for the function of the protein.

• Some proteins require a non-protein molecule to enhance the function or performance of the protein.

– Eg. Hemoglobin requires heme (iron containing compound) to carry the O2

• When a prosthetic group is required by an enzyme it is called a co-enzyme.

– Usually a metal ion or vitamin

• These groups may be covalently or non-covalently linked to the protein.

Multiple Subunit Proteins

• Hemoglobin: – 2 globin subunits.

– 2 globin subunits.

• Non-covalent bonds can form interactions between

individual polypeptide chains: – Binding site – where proteins interact with one another.

– Subunit – each polypeptide chain of large protein.

– Dimer – protein made of 2 subunits. – Can be same subunit or different subunits.

Hemoglobin Model Protein Molecule

Hemoglobin: Background • Protein in red blood cells.

• Composed of four subunits.

• Each subunit contains a heme group.

• A ring-like structure with a central iron atom that binds

oxygen.

– Known as Heme; a prosthetic group.

• Hemoglobin has the function of picking up oxygen from

regions (lungs) of high concentration and delivering it to

regions of low concentrations (active tissues) efficiently.

• Uses “cooperative binding” of the four subunits to sense

if oxygen should be picked or delivered.

– Contains “allosteric sites” that can alter structural conformation

and hence functions.

Hemoglobin Primary Structure

N3H+-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-

Val-Thr-Ala-Leu-Trp-Gly-Lys-Val-Asn-Val-Asp-

Glu-Val-Gly-Gly-Glu-…..COO-

• Beta subunit amino acid sequence.

Hemoglobin Tertiary Structure

• Tertiary structure for one beta subunit. – Containing 8 alpha helices.

Hemoglobin Quaternary Structure

Two alpha subunits and two beta subunits.

(141 Aas per alpha subunit, 146 Aas per beta subunit).

Tertiary Structure Stabilizing

Interactions

• Noncovalent. – Van der Waals forces.

– Transient, weak electrical attraction of one atom for another.

– Hydrophobic.

– Clustering of nonpolar groups.

– Hydrogen bonding.

Hemoglobin The Structure-Function Relationship

Chemical Binding of Hemoglobin & Oxygen

• Hemoglobin combines reversibly with O2.

– Hemoglobin is the unoxygenated form.

– Oxyhemoglobin is when O2 is bound.

• Association and dissociation of Hb & O2 occurs within milliseconds. – Critically fast reaction important for O2 exchange.

– Very loose coordination bonds between Fe2+ and O2, easily reversible.

– Oxygen carried in molecular state (O2) not ionic O2-.

• Sigmoid shaped HbO2 equilibrium curve – Describes molecular reaction between the 4 heme groups.

– Describes heme group’s O2 binding capacity enhancement.

Oxygen Saturation & Capacity

• Up to four oxygen molecules can bind to one

hemoglobin (Hb).

• Ratio of oxygen bound to Hb compared to total

amount that can be bound is Oxygen Saturation.

• Maximal amount of O2 bound to Hb is defined as the

Oxygen Capacity.

Oxygen Binding to Hemoglobin • The oxygen saturation curve of Hb is SIGMOIDAL (S-

shaped). – Result of cooperative binding ("cooperativity"), as a result of

"communication" between different ligand binding sites on the same multimeric protein molecule.

• Initially, Hb is in a low affinity T-state.

• Binding of oxygen causes conformational change in Hb, converting it to the high affinity R-state. – sigmoid saturation curve a composite of:

– A low affinity curve at low oxygen concentrations and

– A high affinity curve at high oxygen concentrations.

• Hemoglobin an example of an allosteric protein. – binding of a ligand to one site on the (multi-subunit) protein

affects the binding properties of another site on the same protein molecule

• Because hemoglobin has four subunits, its binding of oxygen reflects multiple equilibria. – Which results in a sigmoidal curve.

Hemoglobin: Oxygen binding Curve

• Fractional saturation versus the concentration of

oxygen. – Indicates the presence of binding sites that have oxygen.

– The concentration of oxygen is determined by partial pressure.

• Hemoglobin's oxygen-binding curve forms in the

shape of a sigmoidal curve due to the cooperativity of

the hemoglobin subunits.

• As hemoglobin travels from the lungs to the tissues,

the pH in surroundings decreases, and the amount of

CO2 increases. » This causes the hemoglobin to lose it's affinity for oxygen.

» Therefore dropping the oxygen into the tissues.

• Results in the sigmoidal curve for hemoglobin in the

oxygen-binding curve – due to subunit cooperativity.

Hemoglobin: Oxygen Binding Curve

Sequence Similarity, Protein Primary Structure & Function

• Sequence similarity in primary structure implies

protein structural, functional & evolutionary relations. ‒ Proteins of same family have similar functions & structure.

‒ They vary in primary structure but retain certain signature motiffs.

• Low sequence similarity implies little structural

similarity. ‒ Small mutations generally well-tolerated by native structure

but with exceptions!

• An exception - Sickle-cell anemia results from one

residue change in hemoglobin primary structure. ‒ Results in complete loss of protein function.

Sequence Similarity, Protein Primary Structure & Function

• Highly polar (hydrophilic) glutamate is replaced with

nonpolar (hydrophobic) valine.

• Causes hemoglobin molecules to repel water and be

attracted to one another.

• Leads to the formation of long hemoglobin filaments.

• Filaments distort the shape of RBCs - taking “sickle”

shape.

• Rigid structure of sickle cells blocks capillaries and

prevents red blood cells from delivering oxygen.

Hemoglobin & Sickle Cell: Normal Trait • Hemoglobin molecules exist as single, isolated units in

RBC, whether oxygen bound or not.

• Cells maintain basic disc shape, whether transporting oxygen or not.

• Sickle-cell is a result of a single mutation in hemoglobin sequence.

Hemoglobin Polymerization

Normal

Mutant

(Sickle cell anemia)

Protein Denaturation • The disruption of secondary, tertiary and quaternary

protein structures by any of the following:

1. Heat/organics.

- Break apart H bonds and disrupt hydrophobic attractions.

2. Acids/ bases.

- Break H bonds between polar R groups and ionic bonds.

3. Heavy metal ions.

- React with S-S bonds to form solids.

4. Mechanical force.

- Eg. agitation - Stretches chains until bonds break.

Sonication, shearig, grinding depending on the level of

protein organization, eg. extraction from tissue. » Depending on the severity, proteins are able to refold into

functional entities under suitable conditions.

Protein Refolding

Summary • This lecture has introduced you to the basic

structure and functions of proteins and provided

an overview.

• Proteins are molecules of life.

• Functional proteins drive most of the molecular

processes through macromolecular assemblies

with either:

– DNA or

– RNA or

– other proteins or

– As a combination of these.

END