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Chapter 8

Three Dimensional Structures ofProteins

Instructor: Rashid Syed

Textbook: Biochemistry (4th

Edition ) by Donald Voet Judith G. Voet

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Protein Structure

4 levels of protein structure

Primary structure: aa sequence

Secondary structure: regular chainorganization pattern

Tertiary structure: 3D complex folding Quarternary structure: association

between polypeptides

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Primary Structure Amino acid sequence determines primary structure

Unique for each protein; innumerable possibilities

Gene sequence determines aa sequence

Each aa is called a residue; numbering (&synthesis) always from NH 2 end toward COOHend

Amino acids covalently attached to each other byan amide linkage called as a peptide bond.

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Peptide Bond

Peptide bonds are rigid (no free rotation around it) and planar (2 -C and -O=C-N-H- in one plane)

Partial double bond character due to resonance structures of peptide bond (bond length is 1.32 A o instead of 1.49 A o

(single) or 1.27 Ao

(double) Partial sharing of 2 electron pairs between C and N of

peptide bond

Due to steric hindrance, all peptide bonds in proteins are intrans configuration

The 2 bonds around the -carbon have freedom of rotationmaking proteins flexible to bend and fold

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The Peptide Bond is Planar

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Partial Double Bond

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Trans Configuration

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Secondary Structure

Secondary structure is the initial folding pattern(periodic repeats) of the linear polypeptide.

Secondary structure refers to relativearrangement of adjacent amino acids

3 main types of secondary structure: -helix, -

sheet and bend/loop Secondary structures are stabilized by hydrogen

bonds

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The -helix

The -helix is right-handed or clock-wise coil of theaa chain around a central longitudinal axis. (for L-isoforms left-handed helix is not viable due to sterichindrance)

The R groups of aa protrude outward from the helix Each turn has 3.6 aa residues and is 5.4 A o high

The helix is stabilized by H-bonds between N-H and

C=O groups of every 4th

amino acid -helices can wind around each other to form coiled

coils that are extremely stable and found in fibrousstructural proteins such as keratin, myosin (muscle

fibers) etc

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Right-handed -helix

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Right- and Left-handed helices

Structure

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Stability of -helix

Interactions between aa side chains can influence -helix

stability Glu-rich sequence destabilizes -helix at pH 7 because of

repulsion between charges Adjacent lys and / or arg are also unstable A 3-aa separation between unlike charges is ideal for

ionic interaction and imparts stability Bulky aa such as asn, cys, ser in close proximity do not

favor -helix (also because of their shape). Pro and gly do not participate in -helix. Pro has a rigid N-C bond since N is part of a ring. Poly-gly stretchesare more stable in an alternate coiled structure because of

greater flexibility

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Stability of -helix

The peptide bond has a small electricdipole because of it partial double bondcharacter

This imparts a partial + charge to the N-

terminus of the -helix ; and a negativecharge to the C-terminus

The overall helix dipole is enhanced bylack of H-bonding at the 4 terminal aa oneach end

Thus, - charged aa at N-end and +charged aa at C-end have stabilizing

effect

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-Pleated Sheet

Extended stretches of 5 or more aa are called -strands

-strands organized next to each other make -sheets

If adjacent strands are oriented in the same direction (N-endto C-end), it is a parallel -sheet, if adjacent strands run

opposite to each other, it is an antiparallel -sheet. There canalso be mixed -sheets

H-bonding pattern varies depending on type of sheet

R-groups protrude outward from the pleats. Interactions between R-groups influence stability. Gly and ala common

-sheets are usually twisted rather than flat

Fatty acid binding proteins are made almost entirely of -sheets

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-Turn / Bend / Loop

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Relative Probability for Amino Acids to occur inType of Secondary Structure

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Circular dichroism spectra to assess Secondary Structures

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Motifs

Also called supersecondary structure Motifs are very stable folding patterns of

secondary structure.

They are combinations of helices, sheets, bends etc. such motifs are seen repeatedly in many

different proteins. Many kinds of motifs are possible and are

given unique names such as -barrel, zincfingers, leucine zippers etc.

Multiple motifs make up protein domains

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Some Motifs

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-sheets

arranged as a barrel

Common in fattyacid binding

proteins

Motifs

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Tertiary Structure

3-D arrangement of amino acids Polypeptides undergo folding or bundling up.

Includes interactions between amino acids that are

far apart in primary structure The folding creates pockets and sites for binding

substrates, ligands and cofactors

Folding is such that non-polar residues are buriedinside, polar residues are exposed outwards toaqueous environment.

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Tertiary Structure

5 kinds of bonds stabilize tertiary structure: H- bonds, van der waals interactions, hydrophobicinteractions, ionic interactions and disulphidelinkages

In disulphide linkages, the SH groups of twoneighboring cysteines form a S-S- bond called as adisulphide linkage. It is a covalent bond, but readily

cleaved by reducing agents that supply the protons toform the SH groups again

Reducing agents include -mercaptoethanol and DTT

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Reduction of Disulfide Linkages

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Protein Denaturation

The information for every proteins 3-D foldedstructure is inherent in its sequence The naturally occuring pattern of protein folding is

called its native conformation

The unfolding or loss of protein native conformationis called protein denaturation

Changes in temperature, pH, salt concentration and presence of organic solvents or urea and detergents

cause protein denaturation Denaturation is reversible; removal of disrupting

agents cause renaturation. Most proteins renaturespontaneously, others require assistance.

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Co-operativity in Protein Denaturation

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Molecular Chaperones

Sometimes protein folding is facilitated by other proteinscalled molecular chaperones. Chaperones often associate with a polypeptide chain as it is

being synthesized and guide it to fold in the right

functionally active conformation. This usually needs some input of energy in the form of

ATP. Two classes of chaperones: Heat-shock proteins, Hsp70,

are found in cells stressed by heat. They bind to denatured proteins and prevent incorrect folding or aggregation Chaparonins: multi-polypeptide complexes that facilitate

folding by an elaborate mechanism

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Errors in Protein Folding

Various genetic disorders may be the result ofincorrect protein folding

The most common cause of cystic fibrosis is due tomisfolding of the CFTR protein resulting from a

single amino acid deletion in the primary structure.

Mad cow disease and other forms of spongiformencephalopathies, (also called as Prion diseases:

pr oteinaceous infection on ly) result from misfoldingof the Prion Protein (PrP) in the brain. Normallyfolded PrP is converted to misfolded PrP byinteraction with the latter.

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Structure of Myoglobin

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Structural Domains

Many proteins are organized into multiple domains Domains are compact globular units that are connected

by a flexible segment of the polypeptide lackingsecondary/tertiary structure

Domains usually have biological activity. Eachdomain contributes a specific function to the overall

protein

Different proteins may share similar domainstructures, eg: kinase-, cysteine-rich-, globin-domains

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Multi-Domain

Structure ofCa-ATPase

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Domains of c-Src protein

SH3 domain = ~ 60 AASH2 domain = ~100AASH3 domain= ~300 AA kinase domain

(phosphotransferase)

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Fibrous Proteins

Polypeptides chains are arranged in long sheetsor strands

Consist of one predominant secondary structure

Fibrous proteins are structural proteins: theirfunctions include providing shape and

mechanical strength Examples: collagen, -keratin, silk fibrion

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-Keratin

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Collagen

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Silk Fibroin

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Quaternary Structure

Association of more than one polypeptides

Each unit of this protein is called as a subunit andthe protein is an oligomeric protein

Subunits (monomers) can be identical or different The protein is homopolymeric or

heteropolymeric

Disulfide bonds usually stabilize the oligomer.Electrostatic / hydrophobic interactions may alsocontribute to quaternary structure stability.

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Quaternary Structure of Hemoglobin

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Methods for the 3-D Structure of a Protein

NMR spectroscopy and X-ray

crystallography are the two mostcommonly used methods todetermine the 3-D structures of

macromolecules.

Principles of NMR

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Principles of NMR

Nuclei act as small barmagnets and possess a net

magnetic moment

No Magnetic FieldIsotropic conditions

Application ofexternal Bo

B o

Precession aroundexternal Bo

Precession atthe Larmorfrequency

=

Very homogeneousMagnetic field

Principles of NMR

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One-dimensional NMR

Two-dimensional NMR

1H chemical shift

1 5 N c

h e m

i c a l s

h i f t

1H,15N HSQC

Three-dimensional NMR

Principles of NMR

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NMR Spectroscopy

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Wh t l d?

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Why are crystals used?

1. X-ray scattering from a single molecule would be incredibly weak and impossible to detect.

2. A crystal arranges a huge number of moleculesin the same orientation, so that scattered wavescan add up in phase and raise the signal to ameasurable level.

Principles of X ray crystallography

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Principles of X-ray crystallography

X Diff ti

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X-ray Diffraction

1.9

F i th

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Fourier theory

1. The diffraction pattern is related to theobject diffracting the waves through amathematical operation called the

Fourier transform.2. So the real image (electron electron

density) can be obtained by by takingthe Fourier transform as long as boththe amplitude and the phaseinformation is known

What is electron density?

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What is electron density?

A map of the distribution of electrons inthe molecule, i.e . an electron density map.

However, since the electrons are mostlytightly localized around the nuclei, theelectron density map is a pretty good

picture of the molecule.

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Crystallization Process

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Crystallization Process

1. For obtaining crystals, the molecules must

assemble into a periodic lattice.2. Start with a solution of the protein with a fairly high

concentration (2-50 mg/ml) and add reagents thatreduce the solubility close to spontaneousprecipitation (called superstaturated state).

3. With slowing further concentration, and underconditions suitable for the formation of a few

nucleation sites, crystalsmay start to grow.4. Many conditions are generally tested usually by

initial screening, then followed by a systematicoptimization of conditions.

Phase Diagram for Crystallization Experiment

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Phase Diagram for Crystallization Experiment

State A: Protein stays undersaturated and no crystals growState B: Protein crystallizes and the concentration of protein

in solution drops to saturationState C: Protein precipitates, but crystals may still grow

Crystallization Methods

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Crystallization Methods

1. Batch Method2. Vapour Diffusion Method

i) Hanging Dropii) Sitting Dropiii) Sandwich Drop

3. Dialysis Method

Batch Method

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Batch Method

The precipitant and protein are mixed directly

This can be done in a glass vial or under oil

Vapour Diffusion:Hanging Drop Method

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Vapour Diffusion:Hanging Drop Method

Vapour Diffusion: Sitt ing Drop Method

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Vapour Diffusion: Sitt ing Drop Method

Dialysis Method

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Dialysis Method

Protein is equilibrated against a larger volume ofprecipitant through a dialysis membrane.

X-ray crystallography requires a singlel h l

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crystal as the sample

But all you get is precipitate. Need to try more conditions.

X-ray crystallography requires a singlel h l

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crystal as the sample

A good quality crystal is grown for data collection!!!

Protein Structure determination

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Fe

HIS-313

HIS-374

ASP-315

TRP-389

Water

TYR-310

MET-299

TYR-303

Final Model of HIF-PH2 based on X-ray Crystallography

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Final Model of HIF PH2 based on X ray Crystallography

Ribbon Diagram of Crystal Structure of HIF-PH2

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Orderedamino acids188 to 403represented asa ribbondiagram

Mixed / structure with strand corefolded into a

jellyroll motif

Chapter 8

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p

Protein Function: Hemoglobin inMicrocosm

Instructor: Rashid Syed

Textbook: Biochemistry (4 th Edition ) by Donald Voet Judith G. Voet

Protein Function

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Protein Function

Reversible binding of ligands is essential Specificity of ligands and binding sites Ligand binding is often coupled to conformational changes, sometimes

quite dramatic ( Induced Fit )

In multisubunit proteins, conformational changes in one subunit can affectthe others ( Cooperativity )

Interactions can be regulated

Illustrated by :Structures of myoglobin and hemoglobinOxygen-binding curvesRegulation of O 2 binding

F ti f P t i

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Functions of Proteins Storage of ions and molecules

myoglobin, ferritin Transport of ions and molecules

hemoglobin, serotonin transporter Defense against pathogens

antibodies, cytokines Muscle contraction

actin, myosin Biological catalysis

chymotrypsin, lysozyme

Prosthetic Groups

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Prosthetic Groups

Some proteins have non-peptide components as part of the functional unit. These non-peptidecomponents are called as prosthetic groups.

Prosthetic groups are permanently associated withthe protein and are required for protein function

Proteins with prosthetic groups are called

complex or conjugated proteins. otherwise theyare simple proteins.

Ligands

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Ligands

A molecule that binds reversibly to a protein is calledits ligand. Ligands may be small peptides or non-

peptide molecules. Physiological ligands are usuallyendocrine, metabolic or environmental factors.

Ligand binding is a trigger for the activation of proteinfunction.

Ligand binding usually results in a conformationalchange in the proteins 3-D structure.

The ligand binding site on the protein iscomplementary to the ligand in size, shape and charge.

Ligand binding is specific.

Binding: Quantitative Description

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g Q p

Consider a process in which a ligand (L) binds reversibly to a site in the protein (P)

The kinetics of such a process is described by:the association rate constant k a

the dissociation rate constant k d

After some time, the process will reach the equilibrium

where the association and dissociation rates are equal The equilibrium composition is characterized by the the

equilibrium constant K a

+ k a

k d PLPL

d

aa

k

k K ==

]L[]P[

]PL[

]PL[]L[]P[ d a k k =

ak

K ==]PL[

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Think of this as % of ligand binding sites occupied.

100%

50%

% ligand binding sites occupied = = --------------------[L] n

[L] n + K d

How does Le Chatliers Principle apply?

d

ak ]L[]P[

Few ligands, Equilibrium not pushed

very far to right

Lots of ligands, equilibrium pushed very far to the right

Hyperbolic Curve for Ligand Binding

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Hyperbolic Curve for Ligand Binding

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Oxygen binding curve of Myoglobin

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Oxygen-binding curve of Myoglobin

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Structure of Heme

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St uctu e o e e

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Interactionsof aa withheme in

myoglobin


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The primary structure is its sequence. This consists of 153

aa with M r = 16,700 Secondary structure: 8 alpha-helical regions designated as

A to H starting at the NH 2 terminus. No -sheets. The tertiary structure: folding of the 8 helices into a single

domain called the globin fold. Several bends and loops formed in the process; they are

labeled reflecting the helical segments they connect. Folding results in the formation of a pocket or box where

the heme group fits in. Hydrophobic aa are buried in the

interior, lining the heme pocket. The tertiary structure is stabilized mostly by hydrophobic

interactions. There are no disulphide bonds. Myoglobin is monomeric, there is no quarternary structure.



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Hemoglobin subunits are structurallyl l b

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Similar to Myoglobin

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Hemoglobin Structure

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g Hemoglobin is an oligomeric protein made up of 2

dimers, a total of 4 polypeptide chains: 11 22. The (141 aa) and (146 aa) subunits are highly

homologous. Total M r of hemoglobin is 64,500

Each subunit consists of 7 ( ) or 8 ( ) alpha helices andseveral bends and loops folded into a single globindomain.

Each chain has one side where nonpolar groups areexposed rather than polar groups. These regions interact

such that the 4 chains are held together by hydrophobicinteractions. H-bonds and ionic interactions alsocontribute to quarternary structure.

Each subunit has a heme-binding pocket.

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Different forms of Hemoglobin

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Different forms of Hemoglobin

When hemoglobin is bound to O 2, it is calledoxyhemoglobin. This is the relaxed (R ) state.

The form with a vacant O 2 binding site is called

deoxy-hemoglobin and corresponds to the tense (T)state.

If iron is in the oxidized state as Fe +3, it is unable to bind O 2 and this form is called as methemoglobin

CO and NO have higher affinity for heme Fe +2 thanO2 and can displace O 2 from Hb, accounting for theirtoxicity.

Changes Induced by O 2 Binding

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g y 2 g

O 2 binding rearranges electrons within Fe making it morecompact so that it fits snugly within the plane of porphyrin.

Since Fe is bound to histidine of the globin domain, when Femoves, the entire subunit undergoes a conformational

change. This causes hemoglobin to transition from the tense (T) state

to the relaxed (R) state.

Inter-subunit interactions influence O 2 binding to all 4subunits resulting in cooperativity

O 2-binding triggers conformational change

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O 2 binding triggers conformational change

Inter-subunit interactions

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Inter subunit interactions

Two alpha and two beta subunits ( 11 22)

Strong interactions between unlike subunits

About 30 aa involved in inter-subunit interactions;

dimer remains intact after disruption with urea In deoxyhemoglobin, the C-terminal His and Asp of

-subunits interact with lys of -subunit. Also argand asp on C-terminus of one -subunit interact with

those of 2nd

-subunit. In oxyhemoglobin, the histidines have shifted to the

interior and can no longer form ion pairs

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T and R states of Hemoglobin

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g

Hemoglobin exists in two major conformationalstates: Relaxed (R ) and Tense (T)

R state has a higher affinity for O 2. In the absence of O

2, T state is more stable; when O

2

binds, R state is more stable, so hemoglobinundergoes a conformational change to the R state.

The structural change involves readjustment of

interactions between subunits. The 11 and 22 dimers rearrange and rotate

approximately 15 degrees with respect to each other

T-state to R-state Transition

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T-state to R-state Transition

O 2-binding kinetics

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4 subunits, so 4 O 2-binding sites

O 2 binding is cooperative meaning that each subsequent O 2 binds with a higher affinity than the previous one

Similarly, when one O 2 is dissociated, the other three willdissociate at a sequentially faster rate.

Due to positive cooperativity, a single molecule is very rarely partially oxygenated.

There is always a combination of oxygenated anddeoxygenated hemoglobin molecules. The percentage ofhemoglobin molecules that remain oxygenated is represented

by its oxygen saturation

O 2-binding curves show hemoglobin saturation as a functionof the partial pressure for O 2.

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SigmoidalCurve forO 2 binding

Oxygen Saturation Curve

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Oxygen Saturation Curve Saturation is maximum at very high O

2 pressure in the

lungs (pO 2 = ~ 100 torr).

As hemoglobin moves to peripheral organs and the O 2 pressure drops (pO 2 = ~20 torr), saturation also drops

allowing O 2 to be supplied to the tissues. Due to co-operative binding of O 2 to hemoglobin, its

oxygen saturation curve is sigmoid.

Such a curve ensures that at lower pO 2, smalldifferences in O 2 pressure result in big changes in O 2 saturation of hemoglobin. This facilitatesdissociation of O 2 in peripheral tissues.

O 2-transfer from Hemoglobin to Myoglobin

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2

The myoglobin curve shows us that only at extremelylow O 2 pressure, there is no binding of O 2 tomyoglobin.

At relatively low pO 2, the saturation shoots up and

there is almost complete saturation, meaning that allmolecules are associated with O 2.

At the pO 2 that myoglobin is fully saturated,hemoglobin is less than half saturated. This facilitatesthe transfer of O 2 from hemoglobin to myoglobin inthe muscle.

Effectors of O 2 binding

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Small molecules that influence the O 2-binding capacity

of hemoglobin are called as effectors (allostericregulation)

Positive or negative effectors; homotropic or heterotropiceffectors

Oxygen is a homotropic positive effector

Positive effectors shift the O 2-binding curve to the left,negative effectors shift the curve to the right

From a physiological view, negative effectors are beneficial since they increase the supply of oxygen to thetissues.

The Bohr Effect

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The regulation of O 2-binding to hemoglobin by H + and CO 2

is called the Bohr effect Both H + and CO 2 are negative effectors of O 2-binding.

Addition of a proton to His imidazole group at C-terminus of-subunit facilitates formation of salt bridge between His andAsp and stabilization of the T quaternary structure ofdeoxyhemoglobin.

CO 2 reduces O 2 affinity by reacting with terminal NH 2 toform negatively charged carbamate groups that form salt

bridges to stabilize deoxyhemoglobin.

Metabolically active tissues need more O 2; they generatemore CO 2 and H + which causes hemoglobin to release its O 2.

Effect of pH on Oxygen -Binding

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p yg g

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2,3-Bisphosphoglycerate

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, p p g y

2,3-Bisphosphoglycerate is a negative effector.

A single 2,3-BPG binds to a central pocket ofdeoxyhemoglobin and stabilizes it by interactingwith three positively charged aa of each -chain.

2,3-BPG is normally present in RBCs and shifts theO2-saturation curve to the right

Thus, 2,3-BPG favors oxygen dissociation and

therefore its supply to tissues In the event of hypoxia, the body adapts by

increasing the concentration of 2,3-BPG in the RBC

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Fetal Hemoglobin

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Fetal hemoglobin has 2 and 2 chains

The chain is 72% identical to the chain.

A His involved in binding to 2,3-BPG is replaced withSer. Thus, fetal Hb has two less + charge than adult Hb.

The binding affinity of fetal hemoglobin for 2,3-BPG issignificantly lower than that of adult hemoglobin

Thus, the O 2 saturation capacity of fetal hemoglobin is

greater than that of adult hemoglobin This allows for the transfer of maternal O 2 to the

developing fetus.

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Sickle-cell anemia is due toi i h l bi

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a mutation in hemoglobin

Glu6 Val in the chain of Hb The new Valine side chain can bind to a

different Hb molecule to form a strand

This sickles the red blood cells Untreated homozygous individuals

generally die in childhood Heterozygous individuals exhibit a

resistance to malaria

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chem 153a week 3

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