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AnnouncementAnnouncement

I am Hyun-Soo Cho, in Biology Department.This course is Molecular Biochemistry,

(4) Grading: (4) Grading: Mid exam (40%) + Final exam (40%) + attendance, reports (20%)

Mid exam (40%) + Final exam (40%) + attendance, reports (20%)

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(6) How to study biochemstry (6) How to study biochemstry Be familiar with English term Motivate yourself why you should study Molecular biochemistry Read text book carefully Ask any questions

Be familiar with English term Motivate yourself why you should study Molecular biochemistry Read text book carefully Ask any questions

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You may see me during this course if you want My office hours: PM 5:00-6:00 on Thursday How: First, Contact me by E-mail or telephone E-mail address: hscho8@yonsei.ac.kr, 2123-5651

Chapter 2

Protein Composition and Structure

Key Properties of Proteins

1. Proteins are linear polymers built of monomer units called amino acids.• The amino acid sequence of a protein dictates its folding process.• The three dimensional structure of a protein determines its

biological function.

The characteristic three dimensional

structure of the beta subunit of E.coli

DNA polymerase complex allows

DNA to be copied during DNA

replication without the replication

machinery dissociating from the DNA.

2. Proteins contain a wide range of functional groups including alcohols,

thiols, thioethers, carboxylic acids, carboxamides, and a variety of basic

groups.Various combinations of functional groups in amino acids enable

broad spectrum of protein function.

3. Proteins can interact with one another and with other biological

macromolecules to form complex assemblies resulting in new

capabilities.

A hexagonal array of

two kinds of protein filaments

in insect flight tissue

(electron micrograph)

Key Properties of Proteins (continued)

4. Some proteins are quite rigid, whereas others display limited flexibility.

• Structural elements are rigid. Why?

(cytoskeletons, connective tissues, etc.)

• Regulatory elements are flexible. Why?

(protein-protein interaction, signal transduction, etc.)

Key Properties of Proteins (continued)

Conformational Changes

of Lactoferrin

upon iron binding

Absolute Configuration : S

Left, Counter-Clockwise

Absolute Configuration : R

Right, Clockwise

Structure and Stereoisomerism of a-Amino Acids

Only L-amino acids are constituents of proteins.

R : functional group (side chain)

C : -carbon (chiral) NH3+ : amino group COO- : carboxyl group

Proteins Are Built from a Repertoire of 20 Amino Acids

Zwitterionic Character of Amino Acids (Dipolarity)

Amino Acid Nomenclature

Proteins are built from a repertoire of 20 amino acids in all species.

Twenty kinds of side chains vary in size, shape, charge, hydrogen bonding

capacity, hydrophobic character, and chemical reactivity.

Classification of Amino AcidsBased on the Characteristics of Functional Groups

Ala, Gly, Ile,

Leu, Met, Phe,

Pro, Val

Arg, Asn, Asp, Cys,

Gln, Glu, His, Lys,

Ser, Thr, Trp, Tyr

Non Polar (Hydrophobic)Polar (Hydrophilic)

Arg, Lys, HisAsp, Glu

Basic (positive charge)Acidic (negative charge)

Most of the restPhe, Tyr, Trp

Aliphatic (linear chain)Aromatic (ring)

achiral(non-chiral)

Most

Simple

Amino

Acids

MostTypical

AliphaticAminoAcids

MostTypical

Non-PolarHydrophobic

AminoAcids

IsoleucineContains

an AdditionalChiral

Carbon

The side chain of proline is bonded toboth the nitrogen and -carbon atom.

Proline is an imino acid.

Structural flexibility is much more restricted than other amino acids.

Proline markedly influences protein architecture.

MostTypical

AromaticAminoAcids

The hydroxyl groupin tyrosine is

chemically reactive.

Tryptophan containsan indole ring.

The aromatic rings ofTyr and Trp contain

delocalized electronsabsorbing UV light.

Tryptophan and Tyrosine Can Be Usefulfor the Determination of Protein Concentration

Beer’s Law : A = clA : Absorbance,

: extinction coefficient (M-1cm-1),

c : concentration (M),

l : length of light pass (cm)

Maximum absorbance

at 276 nm for Tyrosine

at 280 nm for Tryptophan

Ser and ThrContainAliphatic

Hydroxyl Group.

Ser is morehydrophilic than Ala.

Thr is morehydrophilic than Val.

ThreonineContains

an AdditionalChiral

Carbon

Cysteine is structurally similar to serine but contains

a sulfhydryl, thiol (-SH), group in place of the hydroxyl (-OH) group.

Pairs of sulfhydryl groups can form a disulfide bonds

which can be critical in stabilizing three dimensional structure in some proteins.

Very Polar, Highly Hydrophilic, andPositively Charged Amino Acids

Histidine Ionization

Lys : -amino group

Arg : guanidium group

His : imidazole group

Very Polar,

Highly Hydrophilic,

and

Negatively Charged

Amino Acids

Aspartate : -carboxyl group

Glutamate : -carboxyl group

Asparagine (Asn)

Uncharged Derivatives of Aspartate

-carboxamide group

Glutamine (Gln)

Uncharged Derivatives of Glutamate

-carboxamide group

Ka, acid dissociation constant

The equilibrium constant in acid-base reactions

HA A- + H+

pH and pKa ?

Seven Amino Acids

Containing

Readily Ionizable

Side Chains

Aspartate

Glutamate

Histidine

Cysteine

Tyrosine

Lysine

Arginine

pKa values of

functional groups

in actual proteins

can be dramatically

changed by the

microenvironment

where

the given side chains

are located !!!

Amino Acids Are Linked by Peptide (Amide) Bonds

to Form Polypeptide Chains (Proteins)

• The formation of a peptide bond requires an input of free energy

• But, the peptide bond is very stable once it is formed.

(T1/2 in aqueous solution : 1000 years, hydrolysis rate is so slow)

• The order (sequence) of amino acids in a polypeptide chain is

called the primary structure of a protein.

FirstAmino Acid

SecondAmino Acid

Directionality of Polypeptide ChainN-terminus C-terminus

(YGGFL ≠ LFGGY)

Backbone or Main Chain(Regularly Repeating Part)

vs.Functional Group or Side Chain

(Variable Part)

One Amino Acid in a ProteinIs Called as a Residue.

Alternative Positioning ofthe Oxygen and the Hydrogen

in One Peptide Bond

Alternative Positioning ofthe Oxygen and the Hydrogen

between Neighboring Peptide Bonds

Alternative Positioning ofthe Functional Groups

Between Neighboring Residues

Formation of Polypeptide Chain

Disulfide Bonding

• In some proteins, the linear polypeptide chain can be cross-linked and the most

common cross-links are disulfide bonds between cysteine residues.

• Extracellular proteins form disulfide bonds more often than intracellular

proteins. Why?

• Most natural polypeptide chains contain between 50 and 2000

amino acid residues and are commonly referred to as proteins.

• Less than 50 amino acids oligopeptides or peptides

• The average molecular weight of an amino acid is about 110

Dalton. Thus, the molecular weights of most proteins range

between 5500 and 220000 dalton (i.e. 5.5 kd to 220 kd).

Size of Polypeptide Chain

• Each protein has a unique and precisely defined amino acid sequence.

• Central Dogma : DNA RNA Protein

• Amino acid sequence of a protein determines its structure,

function, and the mechanism of biological action.

• Changes in amino acid sequences Disease, Genetic Engineering

Amino Acid Sequences of Proteins

Chemical Properties of Peptide Bonds

Peptide bonds are planar

Typical Bond Lengthswithin a Peptide Bond

The peptide bonds contain• Considerable double bond character• High H-bond forming capacity to

proteins.

(Peptide Bond Peptide Bond, or

Peptide Bonds Functional Groups)

BUT,

still uncharged tightly packed structure

?

Configurational Properties of Peptide Bonds

Trans-Configuration of -Carbons around a Normal Peptide Bond

Balanced Configuration of -Carbons in X-Pro linkages

Rotational Properties of Peptide Bonds

: the angle of rotation about the bond between the nitrogen and the -carbon

: the angle of rotation about the -carbon and the carbonyl carbon

Peptide bonds are rigid…But,

the bonds containing the -carbon between two peptide bonds

can be rotated from -180o to +180o.

Rotational Properties of Peptide Bonds (continued)

• Ramachandran Diagram Shows the Allowed Ranges of and Rotations.

• For Some Combinations of and Rotations Are Physically Impossible due to Steric

Clashes.

• Protein folding is possible by rigidity of peptide unit and restriction of and Rotations

Proteins’ Secondary Structures

Alpha Helix, Beta Pleated Sheet, Turns, Loops

Linus Pauling and Robert Corey’s proposal – 1951The Double helix – 1953 James D. Watson, 책소개

The -Helix Is a Coiled StructureStabilized by Intra-Chain Hydrogen Bonds

• Rise of 1.5 Å per Residue

along the Helix Axis

• Rotation of 100 degree per

Residue around the Helical

Turning

• 3.6 Amino Acids per a Single

Turn of -Helix

• Thus, amino acids spaced

three to four residues apart

are spatially quite close to

one another in an -helix.

Most plausible H-bondingsbetween peptide bonds in -helix

(n, n+4)

• Essentially all -helices in proteins are right

handed.

(Ramachandran diagram explains it why.)

• The -helical content of proteins range from none

to almost 100%.

• Single -helices are usually less than 45 Å long.

• Two or more helices can be entwined and form a

very stable and long coiled coil structure with a

length of 1000 Å long

Ferritin contains 75% of -helices.

-helical coiled coil; superhelix;tropomyosin, keratin, fibrin;

bundles of fibers; filamentous structures

Probability of-Helix Coiling Direction

Coiling and Entwining of -Helix

Beta Pleated Sheet

Combinations of and rotationsallowing the formation of -sheet

• Almost Fully Extended Structure

• Distance between Amino acids is 3.5 Å.

• The side chains of adjacent amino acids point

in opposite directions.

• H-bonding between different -strands

• Why beta?

Anti-Parallel -Sheet

H-Bondings

between

Single Amino Acids

Parallel -Sheet

Overlapped H-Bondings

between

Two Amino Acids

Two Simplest -Sheet Structures

More -Sheet Structures

Mixed -Sheet Structurewith Multiple -Strands

Twisted -Sheet Structurewith Multiple -Strands

An Example ofa Protein

Rich in -Sheet

Fatty AcidBinding Protein

Turns and Loops

• Reverse Turn (-turn, hairpin bend)

enables reversals in the direction of

polypeptide chains.• These reversals allow proteins to form

compact and globular structures.

• Omega Loop ( loop) also

enables reversals in the direction of

polypeptide chains.• No regular and periodic structures• But, loop structures could also be

rigid and well defined.• Invariably located on the surface• Protein-protein interactions

Coiled-coil protein

• Structural support for Cells and Tissues

-keratin: left-handed superhelix of two right-handed helices.

from wool & hair, intermediate filaments in cytoskeleton, muscle protein

(myosin & tropomyosin)

Heptad repeats; Every seventh residue in each helix, Leu holds two helix

by van der Waals interactions Why 7?

disulfide bond crosslinks: fewer – flexible, more – harder (horns, claws etc)

•Collagen: the most abundant protein of mammals, main fibrous

component of skin, bone, tendon, cartilage, and teeth. ( 피부미용 )

What is Van der Waals force?

Weak electric forces between neutral molecules by flucutating polarization of nearby particles

3 source; permanent dipole-permanent dipole forces, permanent dipole-induced dipole force, Instantaneous induced dipole-induced dipole (London dispersion forces)

Named after Dutch physicist, Johannes Diderik van der Waals Nobel prize winner in physics in 1910

van der waals interaction

-caused by transient dipoles, the momentary random fluctuation in the distribution of the electrons of any atoms- 1/r6 dependence

Figure 1-10 Table of the typical chemical interactions that stabilize polypeptides

1-4. Bonds that Stabilize Folded Proteins

1 kcal = 4.2 kJ

Folded proteins are stabilized mainly by weak noncovalent interactions

Tertiary Structure

• Water-soluble proteins fold into

compact structure with nonpolar

cores

• Three dimensional structure of a

polypeptide chain – grouping of

amino acids

• Generally, protein folding yields very

compact tertiary structures (10 fold).

3D Structure of Myoglobin

1) 7 -helices (70% of main chain) are linked by turns and loops.

2) Heme = Protoporphyrin + Iron; Prosthetic Group; Oxygen Binding

• The interior space consists almost entirely of non-polar residues.

(e.g. Val, Leu, Met, Phe, etc.)

• The charged residues are absent from the inside of a protein.

(e.g. Asp, Glu, Lys, Arg, etc.)

• The only polar residues inside are two His; iron and oxygen binding

• The surface outside consists of both polar and non-polar residues.

• There is very little free empty space inside.

Key Aspects of Myoglobin 3D Structure

Surface cross section

General Rules of Protein Folding

• In an aqueous environment, protein folding is driven by the strong

tendency of hydrophobic residues to be excluded from water.- called

hydrophobic effect, Why?

• Contrasting distribution of polar and non-polar residues: the hydrophobic

side chains are buried inside, whereas the hydrophilic and charged

functional groups are headed to the outer surface.

• All the NH and CO groups from the interiorly located peptide bonds

holding non-polar side chains (i.e. peptide bonds around hydrophobic

environment) are forced to form hydrogen bonds.

• Therefore, these multiple hydrogen bonding enhance the interior

structural integrity by efficiently establishing -helix and -sheet

structures.

• Van der Waals interactions between hydrophobic side chains also

contributes to the structural stability of a protein.

Hydrophobic Effects?

Inside-Out Folding : Exception of Protein Folding

Membrane protein, Porin

• Proteins found in the outer membranes of

many bacteria

• The outside is covered with hydrophobic

residues interacting with neighboring alkane

chains. (cf. permeability barriers of the

biological membranes)

• The center of the protein contain a water-

filled channel lined with charged and polar

amino acids.

• Hydrophobic vs. Aqueous Environment

• Membrane vs. Cytosolic Proteins

4 Domains in CD4 ; Each domain with approximately 100 Amino Acids

• A compact and globular structural unit of a protein is often called as a domain

(i.e. pearls on a string)

• The size of a domain ranges from 30 to 400 amino acid residues.

• Different proteins can have a similar or the same domain.

• Domain is a structural working unit of a protein for the common function.

Domain

Motif

• Functional supersecondary structure

•DNA binding proteins

Four level of structural organization

•Primary structure: the amino acid sequence

•Secondary structure: spatial arrangement of a.a nearby in sequence, helix and strand

•Tertiary structure: spatial arrangement of a.a far apart in sequence.

Quaternary Structure

The spatial arrangement of subunits and the nature of their interaction

Subunit

each polypeptide chain in a protein containing more than one polypeptide chain

Cro(Bacteriophage )

a dimer ofidentical subunits

(homodimer)

HemoglobinHetero - Tetramer

(22)

RhinovirusCoat Protein

60 copies of eachof 4 subunits

Common cold

Ribonuclease (124 AA; 4 Disulfide Bonds) Denaturant

Reductant

The Amino Acid Sequence of a protein Determines Its Three-Dimenssional Structure

A Lesson from Ribonuclease Observed by Anfinsen

The information needed to specify the catalytically active structure of ribonuclease

is contained in its amino acid sequence : Sequence specifies conformation !!!

8M urea

-mercaptoethanol

trace of-mercapto

ethanol

slow dialysis &oxidation

slow refolding & regaining activity

remove -mercaptoethanol first and then remove urea

Random coiled ribonucleasescrambled

VDLLKN in -helix VDLLKN in -strand

Many sequences can adopt alternative conformations

In many cases,

the context is very crucial in determining the conformational outcome.

(cf. the accuracy of predicting secondary structures using oligopeptides < 60 to 70%)

How a.a. sequence specify protein structure?How an unfolded polypeptide chain acquire the native tertiary structure?How about secondary structure?

-Helix could be default.

Val and Ile prefer

-sheet due to

their branching at -carbon.

Pro breaks

both -helices and -sheets

due to its ring structure.

Ser, Asp, Asn often disturb

the formation of

-helix due to their

capability

to easily form

extra hydrogen bonds

with various side chains.

Each amino acid has its own preference

to form -helix, -sheet, or turns.

Protein Misfolding & Aggregation Can Cause Neurological Diseases

Prion Diseases• Bovine Spongiform Encephalopathy (Mad Cow Disease), Creutzfeldt-Jakob Disease

(Human), Scrapie (Sheep); Disease transmitted purely by protein agents termed

“PRION”; Stanley Prusiner 1997 Nobel Prize• Transmissible agents are aggregated fibrous forms of a specific protein.• These protease resistant aggregated proteins are often referred as amyloid forms.• These amyloid fibers are derived from a normal cellular protein, called PrP, in brain.• Structural conversion from -helices to -sheets

Alzheimer Disease and Parkinson Disease• amyloid plaques A (-amyloid peptide) APP (amyloid precursor protein).• Large aggregates not toxic, smaller aggregate damaging cell membrane.

Prions

-amyloid plaques

Cooperativeness in Protein Folding : ALL or NONE Process

1:1 Mixture (Folded & Unfolded Proteins), no half-folded protein

Cooperative and SharpTransition

Computational prediction of folding is not yet reliable

• Ab initio method - Equilibrium conformation is the global free-energy minimum - potential energy parameter is accurate (H-bond, van der Waals

etc)- key intermediates?

- oligomerization can not be addressed although very many globular proteins are oligomeric.

Protein folding funnel

STILL More to Give a Thought……

• Progressive Stabilization of Intermediates during Folding rather than random search

• Prediction of Three Dimensional Structure from Amino Acid Sequence?

- ab initio prediction

- knowledge-based methods

• Post-Translational Modification

- Phosphorylation: serine, threonine, and tyrosine, signaling switch

- Glycosylation: Asn (N) and Ser and Thr (O-GlcNAc), solubility increase and protein-protein

interaction

- Acetylation: N terminal of proteins, resistant to degradation.

- Hydroxylation: hydroxylation of proline in collagen stabilization, Vitamin C deficency

- Carboxylation: glutamate in prothrombin, Vitamin K deficency - hemorrhage

- Acylation: additon of a fatty acid to a-amino group or cysteine sulfhydryl group

-Carbamylation

Nuclear localization of a steroid receptor

(+) corticosterone

Fluorescence of GFP chromophore by cyclization reaction including rearrangement and oxidation

•composed of 238 amino acids (26.9 kDa), originally isolated from the jellyfish Aequorea victoria

•fluorescens green when exposed to blue light

•Used as a reporter of expression & biosensor

•The GFP gene can be introduced into organisms (bacteria, yeast and other fungal cells, plant, fly, and mammalian cells)

•2008 Nobel Prize in Chemistry : Martin Chalfie, Osamu Shimomura and Roger Y. Tsien

•A typical beta barrel structure

Green fluorescent protein (GFP)

Cleavage after protein synthesis - digestive enzymes (pancreas, intestine) - blood clotting factor (fibrinogen firbrin) - hormone, viral proteins

Summary

• Proteins are built from a repertoire of 20 amino acids

• Peptide bond

• Protein structure; four levels - primary structure - secondary structure ( helix, sheet, turns and loop), - tertiary structure - quarternary structure

• protein folding & misfolding or aggregation

• Protein modification

•How to visualize molecular structures using pymol homeworks