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Principles of Protein Structure

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Primary Structure - Amino Acids

• It is the amino acid sequence (1940) that “exclusively” determines the 3D structure of a protein

• 20 amino acids – modifications do occur post translationally

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Amino Acids Continued…

• Chirality – amino acids are enatiomorphs, that is mirror images exist – only the L(S) form is found in naturally forming proteins. Some enzymes can produce D(R) amino acids

• Think about a data structure for this information – annotation and a validation procedure should be included

• Think about systematic versus common nomenclature

Primary Structure

Formation of cystine

Amino acids

• Polar, uncharged amino acids– Contain R-groups that can form hydrogen bonds with water– Includes amino acids with alcohols in R-groups (Ser, Thr, Tyr)– Amide groups: Asn and Gln– Usually more soluble in water

• Exception is Tyr (most insoluble at 0.453 g/L at 25 C)– Sulfhydryl group: Cys

• Cys can form a disulfide bond (2 cysteines can make one cystine)

Amino acids • Acidic amino acids

– Amino acids in which R-group contains a carboxyl group– Asp and Glu– Have a net negative charge at pH 7 (negatively charged pH

> 3)– Negative charges play important roles

• Metal-binding sites• Carboxyl groups may act as nucleophiles in

enzymatic interactions• Electrostatic bonding interactions

Amino acids

• Basic amino acids– Amino acids in which R-group have net positive charges at pH 7– His, Lys, and Arg– Lys and Arg are fully protonated at pH 7

• Participate in electrostatic interactions– His has a side chain pKa of 6.0 and is only 10% protonated at pH 7– Because His has a pKa near neutral, it plays important roles as a proton

donor or acceptor in many enzymes.– His containing peptides are important biological buffers

Nonstandard amino acids

• 20 common amino acids programmed by genetic code• Nature often needs more variation • Nonstandard amino acids play a variety of roles: structural, antibiotics,

signals, hormones, neurotransmitters, intermediates in metabolic cycles, etc.

• Nonstandard amino acids are usually the result of modification of a standard amino acid after a polypeptide has been synthesized.

• If you see the structure, could you tell where these nonstandard amino acids were derived from?

Nonstandard amino acids

Nonstandard amino acids

Peptide bonds • Proteins are sometimes called polypeptides since they contain many peptide bonds

H

C

R1

H3N+

C

O

OH NH

H

C

R2

O-C

OH

+

H

C N

R1

H3N+

C

O

H H

C

R2

O-C

O

+ H2O

Structural character of amide groups • Understanding the chemical character of the amide is important since the peptide bond is an amide bond.• These characteristics are true for the amide containing amino acids as well (Asn, Gln)• Amides will not ionize:

R C

O

NH2 R C

O

NH2

Acid-base properties of amino acids

K1=

Gly+ + H2O Gly0 + H3O+

[Gly0][H3O+][Gly+]

Gly0 + H2O Gly- + H3O+

K2=[Gly-][H3O+]

[Gly0]

The dissociation of first proton from the -carboxyl group is

The dissociation of the second proton from the -amino group

The pKa’s of these two groups are far enough apart that they can be approximated by Henderson-Hasselbalch

pK1 + logpH =[Gly0][Gly+]

pK2 + logpH =[Gly-][Gly0]

Titration curve of glycine

H

C H

COO-

H3N+ Neutral

form

Titration of Gly

H

C H

COO-

H3N+

H

C H

COO-

H2N

H

C H

COOH

H3N+

pK1 pK2

Gly0Gly+ Gly-

pH 2.3 pH 9.6

From the pK values we can calculate the pI (isoelectric point) where the amino acid is neutral.

pI ≈ average of (pK below neutral+ pK above neutral)

So, for Gly, pI = (pK1 + pK2)/2 = (2.3 + 9.6)/2 ≈ 6

General rules for amino acid ionization• Alpha carboxylic acids ionize at acidic pH and have pKs less than 6;

So in titrating a fully protonated amino acid, alpha carboxylic acids lose the proton first.

• Alpha amino groups ionize at basic pH and have pKs greater than 8; So after acids lose their protons, amino groups lose their proton.

• Most of the 20 amino acids are similar to Gly in their ionization properties because their side chains do not ionize at biological pHs.

• However, there are 5 exceptions worth noting (the amino acids with polar charged side chains)

• Glu, Asp, Lys, Arg, His• Each has 3 ionizible groups and thus, 3 pKs.

Titration curve of arginine

The neutral form of Asp is close to pH 10.8Take the pKs for +1 and -1 from this point and average to get approximate pI,pI = (pK2 + pK3)/2 = (9.0 + 13.0)/2 = 11.0

Acid-base properties of amino acids Amino acid -COOH pKa -NH3

+ pKa R-group pKa

Gly 2.3 9.6 -

Ala 2.4 9.7 -

Val 2.3 9.6 -

Leu 2.4 9.6 -

Iso 2.4 9.7 -

Met 2.4 9.2 -

Pro 2.1 10.6 -

Phe 1.8 9.1 -

Trp 2.4 9.4 -

Ser 2.2 9.2 13

Thr 2.6 10.4 13

Tyr 2.2 9.1 10.1

Cys 1.7 10.8 8.3

Asn 2.0 8.8 -

Gln 2.2 9.1 -

Asp 2.1 9.8 3.9

Glu 2.2 9.7 4.3

Lys 2.2 9.0 10.5

Arg 2.2 9.0 12.5

His 2.4 9.2 6.0

More rules for amino acid ionization• Carboxylic acid groups near an amino group in a molecule have a more acidic

pK than isolated carboxylic groups.

• Amino groups near a carboxylic acid group also have a more acidic pK than isolated amines.

• Aromatic amines like His have a pK about pH 6.

• When titrating an amino acid that is fully protonated (ie starting at pH = 1), the alpha carboxylic acids lose their proton first (all free amino acids have this group), then side chain carboxylic acids, then aromatic amine side chains (His), then alpha amino groups, then side chain amino groups.

• These rules apply to small peptides too.

Amino acids are optically active

• All amino acids are optically active (exception Gly).• Optically active molecules have asymmetry; not superimposable (mirror images)• Central atoms are chiral centers or asymmetric centers. • Enantiomers -molecules that are nonsuperimposable mirror images

Asymmetry• Molecules are classified as Dextrorotatory (right handed), D or

Levrotatory (left handed) L depending on whether they rotate the plane of plane-polarized light clockwise or counterclockwise determined by a polarimeter

Asymmetry• Fischer projections are a shorthand way to write molecules with chiral

centers

Asymmetry• All -amino acids from proteins have the L-stereochemical

configuration

Diastereomers• Stereoisomers or optical isomers are molecules with different

configurations about at least one of their chiral centers but are otherwise identical

• Since each asymmetric center in a chiral molecule can have two possible configurations, a molecule with n chiral centers has 2n different possible stereoisomers and 2n-1 enantiomeric pairs

• Ex. Threonine and Isoleucine both have two chiral centers, and thus 4 possible stereoisomers.

Diastereomers

*

*

Diastereomers• Special case: 2 asymmetric centers are chemically identical (2

asymmetric centers are mirror images of one another)• A molecule that is superimposable on its mirror image is optically

inactive (meso form)

Nomenclature • Glx can be Glu or Gln• Asx can be Asp or Asn• Polypeptide chains are always described from the N-terminus to the C-

terminus

Nomenclature • Nonhydrogen atoms of the amino acid side chain are named in sequence

with the Greek alphabet

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Peptide Bond Formation• Individual amino acids form a polypeptide chain• Such a chain is a component of a hierarchy for describing

macromolecular structure• The chain has its own set of attributes• The peptide linkage is planar and rigid

Primary Structure

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Geometry of the Chain• A dihedral angle is the angle

between two planes defined by 4 atoms – 123 make one plane; 234 the other

• Omega is the rotation around the peptide bond Cn – Nn+1 – it is planar and is 180 under ideal conditions

• Phi is the angle around N – Calpha

• Psi is the angle around Calpha C’• The values of phi and psi are

constrained to certain values based on steric clashes of the R group. Thus these values show characteristic patterns as defined by the Ramachandran plot

From Brandon and ToozeSecondary Structure

Dihedral Angles

Properties of alpha helix

• 3.6 residues per turn, 13 atoms between H-bond donor and acceptor approx. -60º; approx. -40º• H- bond between C=O of ith residue & -NH of (i+4)th residue• First -NH and last C=O groups at the ends of helices do not participate in H-

bond• Ends of helices are polar, and almost always at surfaces of proteins• Always right- handed• Macro- dipole

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Alpha Helix Continued

• There are 3.6 residues per turn

• A helical wheel will outline the surface properties of the helix

Secondary Structure

Alpha Helix

Introduction to Molecular BiophysicsAssociation of helices: coiled coils

These coiled coils have a heptad repeat abcdefg with nonpolar residues at position a and d and an electrostatic interaction between residues e and g.

Isolated alpha helices are unstable in solution but arevery stable in coiled coil structures because of the interactions between them

The chains in a coiled-coil have the polypeptide chains aligned parallel and in exact axial register. This maximizes coil formation between chains.

The coiled coil is a protein motif that is often used to control oligomerization.

They involve a number of alpha-helices wound around each other in a highly organised manner, similar to the strands of a rope.

Introduction to Molecular BiophysicsThe Leucine Zipper Coiled Coil

Initially identified as a structural motif in proteins involved in eukaryotic transcription. (Landschultz et al., Science 240: 1759-1763 (1988). Important part of Eugenetics. Originally identified in the liver transcription factor C/EBP which has a Leu at every seventh position in a 28 residue segment.

Association of helices: coiled coilsThe helices do not have to run in the same direction for this type of interaction to occur, although parallel conformation is more common.

Antiparallel conformation is very rare in trimers and unknown in pentamers, but more common in intramolecular dimers, where the two helices are often connected by a short loop.

Chan et al., Cell 89, Pages 263-273.

Since the dipole moment of a peptide bond is 3.5 Debye units, the alpha helix has a net macrodipole of:

n X 3.5 Debye units (where n= number of residues)

This is equivalent to 0.5 – 0.7 unit charge at the end of the helix.

Basis for the helical dipoleIn an alpha helix all of the peptidedipoles are oriented along the same direction.

Consequently, the alpha helix has a net dipole moment.

The amino terminus of an alpha helix is positive and the carboxy terminus is negative.

Common Secondary Structure Elements

• The Beta Sheet

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Beta Sheets

Secondary Structure

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Beta Sheets Continued• Between adjacent polypeptide chains• Phi and psi are rotated approximately 180 degrees from

each other• Mixed sheets are less common• Viewed end on the sheet has a right handed twist that may

fold back upon itself leading to a barrel shape (a beta barrel)

• Beta bulge is a variant; residue on one strand forms two hydrogen bonds with residue on other – causes one strand to bulge – occurs most frequently in parallel sheets

Secondary Structure

Secondary structure: reverse turns

Secondary Structure:Phi & Psi Angles Defined

• Rotational constraints emerge from interactions with bulky groups (ie. side chains).

• Phi & Psi angles define the secondary structure adopted by a protein.

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Other Secondary Structures – Loop or Coil

• Often functionally significant• Different types

– Hairpin loops (aka reverse turns) – often between anti-parallel beta strands

– Omega loops – beginning and end close (6-16 residues)

– Extended loops – more than 16 residues

Secondary Structure

1AKK

The dihedral angles at C atom of every residue provide polypeptides requisite conformational

diversity, whereby the polypeptide chain can fold into a globular shape

Ramachandran Plot

Structure Phi () Psi()Antiparallel -sheet -139 +135Parallel -Sheet -119 +113Right-handed -helix +64 +40310 helix -49 -26 helix -57 -70Polyproline I -83 +158Polyproline II -78 +149Polyglycine II -80 +150

Phi & Psi angles for Regular Secondary Structure Conformations

Table 10

Secondary Structure

Beyond Secondary StructureBeyond Secondary Structure

Supersecondary structure (motifs): small, discrete, commonly observed aggregates of secondary structures

sheet helix-loop-helix

Domains: independent units of structure barrel four-helix bundle

*Domains and motifs sometimes interchanged*

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Secondary Structure

• The chemical nature of the carboxyl and amino groups of all amino acids permit hydrogen bond formation (stability) and hence defines secondary structures within the protein.

• The R group has an impact on the likelihood of secondary structure formation (proline is an extreme case)

• This leads to a propensity for amino acids to exist in a particular secondary structure conformation

• Helices and sheets are the regular secondary structures, but irregular secondary structures exist and can be critical for biological function

Secondary Structure

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Other (Rarer) Helix Types - 310

• Less favorable geometry

• 3 residues per turn with i+3 not i+4

• Hence narrower and more elongated

• Usually seen at the end of an alpha helix

Secondary Structure 4HHB

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Other (Very Rare) Helix Types - Π

• Less favorable geometry• 4 residues per turn with i+5 not i+4• Squat and constrained

Secondary Structure

Supersecondary structure: Crossovers in ---motifs

Right handed

Left handed

• Consists of two perpendicular 10 to 12 residue alpha helices with a 12-residue loop region between

• Form a single calcium-binding site (helix-loop-helix). • Calcium ions interact with residues contained within the loop

region. • Each of the 12 residues in the loop region is important for

calcium coordination. • In most EF-hand proteins the residue at position 12 is a

glutamate. The glutamate contributes both its side-chain oxygens for calcium coordination.

EF Hand

Calmodulin, recoverin : Regulatory proteins Calbindin, parvalbumin: Structural proteins

EF Fold

Found in Calcium binding proteins such as Calmodulin

•Consists of two helices and a short extended amino acid chain between them.  •Carboxyl-terminal helix fits into the major groove of DNA.   •This motif is found in DNA-binding proteins, including repressor, tryptophan repressor, catabolite activator protein (CAP)

Helix Turn Helix Motif

Leucine Zipper

•The beta-alpha-beta-alpha-beta subunit•Often present in nucleotide-binding proteins

Rossman Fold

What is a Protein Fold? Compact, globular folding arrangement of the polypeptide chain

Chain folds to optimise packing of the hydrophobic residues in the interior core of the protein

Common folds

Tertiary structure examples: All-

AlamethicinThe lone helix

Rophelix-turn-helix

Cytochrome Cfour-helix bundle

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Tertiary Structure

• Myoglobin (Kendrew 1958) and hemoglobin (Perutz 1960) gave us the proven experimental insights into tertiary structure as secondary structures interacting by a variety of mechanisms

• While backbone interactions define most of the secondary structure interactions, it is the side chains that define the tertiary interactions

Tertiary Structure

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Components of Tertiary Structure

• Fold – used differently in different contexts – most broadly a reproducible and recognizable 3 dimensional arrangement

• Domain – a compact and self folding component of the protein that usually represents a discreet structural and functional unit

• Motif (aka supersecondary structure) a recognizable subcomponent of the fold – several motifs usually comprise a domain

Like all fields these terms are not used strictly making capturing data that conforms to these terms all the more difficult

Tertiary Structure

Domains• A domaindomain 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

• Different domains can impart different functions to proteins

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

Domains

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Tertiary Structure as Dictated by the Environment

• Proteins exist in an aqueous environment where hydrophilic residues tend to group at the surface and hydrophobic residues form the core – but the backbone of all residues is somewhat hydrophilic – therefore it is important to have this neutralized by satisfying all hydrogen bonds as is achieved in the formation of secondary structures

• Polar residues must be satisfied in the same way – on occasion pockets of water (discreet from the solvent) exist as an intrinsic part of the protein to satisfy this need

• Ion pairs (aka salt bridge) form important interactions

• Disulphide linkages between cysteines form the strongest (ie covalent tertiary linkages); the majority of cysteines do not form such linkages

Tertiary Structure

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Tertiary Structure as Dictated by Protein Modification

• To the amino acid itself eg hydroxyproline needed for collagen formation

• Addition of carbohydrates (intracellular localization)

• Addition of lipids (binding to the membrane)

• Association with small molecules – notably metals eg hemoglobin

Tertiary Structure

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There are Different Forms of Classification apart from Structural

• Biochemical– Globular – Membrane– Fibrous

myoglobin

Collagen

Bacteriorhodopsin

Tertiary structure examples: All-

sandwich barrel

Tertiary structure examples:

placental ribonucleaseinhibitor horseshoe

triose phosphateisomerase barrel

Four helix bundle

•24 amino acid peptide with a hydrophobic surface•Assembles into 4 helix bundle through hydrophobic regions•Maintains solubility of membrane proteins

TIM Barrel

•The eight-stranded / barrel (TIM barrel)

•The most common tertiary fold observed in high resolution protein crystal structures

•10% of all known enzymes have this domain

Zinc Finger Motif

Domains are independently folding structural units.

Often, but not necessarily, they are contiguous on the peptide chain. Often domain boundaries are also intron boundaries.

Domain swapping: Parts of a peptide chain can reach into neighboring

structural elements: helices/strands in other domains or whole domains in other subunits.

Domain swapped diphteria toxin:

• Helix bundlesLong stretches of apolar amino acidsFold into transmembrane alpha-helices“Positive-inside rule”

Cell surface receptorsIon channelsActive and passive transporters

• Beta-barrelAnti-parallel sheets rolled into cylinder Outer membrane of Gram-negative bacteria

Porins (passive, selective diffusion)

Transmembrane Motifs

Quaternary Structure

• Refers to the organization of subunits in a protein with multiple subunits

• Subunits may be identical or different

• Subunits have a defined stoichiometry and arrangement

• Subunits held together by weak, noncovalent interactions (hydrophobic, electrostatic)

• Associate to form dimers, trimers, tetramers etc. (oligomer)

• Typical Kd for two subunits: 10-8 to 10-16M (tight association)–Entropy loss due to association - unfavorable –Entropy gain due to burying of hydrophobic groups - very favourable

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Quaternary Structure

• The biological function of some molecules is determined by multiple polypeptide chains – multimeric proteins

• Chains can be identical eg homeodimer or different eg heterodimer

• The interactions within multimers is the same as that found in tertiary and secondary structures

• Stability: reduction of surface to volume ratio • Genetic economy and efficiency • Bringing catalytic sites together • Cooperativity (allostery)

Structural and functional advantages of quaternary structure

Quaternary structure ofmultidomain proteins

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Cooperativity

Co-location of Function

Combination

Structural Assembly

Hemoglobin:Enhanced bindingcapability of oxygen

Glutamine sythetase:Controlled use ofNitrogen from Multiple active sites

Immunoglobulin:Multiple receptorresponses

Actin:Giving the cell shape and form

Quaternary Structure

Useful Proteins

• There are thousands and thousands of different combinations of amino acids that can make up proteins and that would increase if each one had multiple shapes

• Proteins usually have only one useful conformation because otherwise it would not be efficient use of the energy available to the system

• Natural selection has eliminated proteins that do not perform a specific function in the cell

Protein Families

• Have similarities in amino acid sequence and 3-D structure

• Have similar functions such as breakdown proteins but do it differently

Proteins – Multiple Peptides

• 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

Single Subunit Proteins

Different Subunit Proteins

• Hemoglobin– 2 globin

subunits– 2 globin

subunits

Protein Assemblies• Proteins can form very

large assemblies• Can form long chains if

the protein has 2 binding sites – link together as a helix or a ring

• Actin fibers in muscles and cytoskeleton – is made from thousands of actin molecules as a helical fiber

Types of Proteins

• Globular ProteinsGlobular Proteins – most of what we have dealt with so far– Compact shape like a ball with irregular

surfaces– Enzymes are globular

• Fibrous ProteinsFibrous Proteins – usually span a long distance in the cell– 3-D structure is usually long and rod shaped

Important Fibrous Proteins• Intermediate filaments of the cytoskeleton

– Structural scaffold inside the cell• Keratin in hair, horns and nails

• 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

• Elastin – unstructured fibers that gives tissue an elastic characteristic

Collagen and Elastin

Stabilizing Cross-Links

• Cross linkages can be between 2 parts of a protein or between 2 subunits

• Disulfide bonds (S-S) form between adjacent -SH groups on the amino acid cysteine

Proteins at Work

• The conformation of a protein gives it a unique function

• To work proteins must interact with other molecules, usually 1 or a few molecules from the thousands to 1 protein

• Ligand – the molecule that a protein can bind• Binding site – part of the protein that interacts

with the ligand– Consists of a cavity formed by a specific arrangement

of amino acids

Ligand Binding

Formation of Binding Site

• The binding site forms when amino acids from within the protein come together in the folding

• The remaining sequences may play a role in regulating the protein’s activity

Antibody Family

• A family of proteins that can be created to bind to almost any molecule

• AntibodiesAntibodies (immunoglobulins) are made in response to a foreign molecule ie. bacteria, virus, pollen… called the antigenantigen

• Bind together tightly and therefore inactivates the antigen or marks it for destruction

Antibodies

• Y-shaped molecules with 2 binding sites at the upper ends of the Y

• The loops of polypeptides on the end of the binding site are what imparts the recognition of the antigen

• Changes in the sequence of the loops make the antibody recognize different antigens - specificity

Antibodies

Binding Strength• Can be measured directly• Antibodies and antigens are mixing around in a

solution, eventually they will bump into each other in a way that the antigen sticks to the antibody, eventually they will separate due to the motion in the molecules

• This process continues until the equilibrium equilibrium is reached – number sticking is constant and number leaving is constant

• This can be determined for any protein and its ligandligand

Equilibrium Constant

• Concentration of antigen, antibody and antigen/antibody complex at equilibrium can be measured – equilibrium equilibrium constant (K)constant (K)

• Larger the K the tighter the binding or the more non-covalent bonds that hold the 2 together

Enzymes as Catalysts

• Enzymes are proteins that bind to their ligand as the 1st step in a process

• An enzyme’s ligand is called a substratesubstrate– May be 1 or more molecules

• Output of the reaction is called the product• Enzymes can repeat these steps many times and

rapidly, called catalysts• Many different kinds – see table 5-2, p 168

Enzymes at Work• Lysozyme is an important enzyme that protects us

from bacteria by making holes in the bacterial cell wall and causing it to break

• Lysozyme adds H2O to the glycosidic bond in the cell wall

• Lysozyme holds the polysaccharide in a position that allows the H2O to break the bond – this is the transition statetransition state – state between substrate and product

• Active siteActive site is a special binding site in enzymes where the chemical reaction takes place

Lysozyme

• Non-covalent bonds hold the polysaccharide in the active site until the reaction occurs

Features of Enzyme Catalysis

Prosthetic Groups• 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 performance of the protein – Hemoglobin requires heme (iron containing compound)

to carry the O2

• When a prosthetic groupprosthetic group is required by an enzyme it is called a co-enzymeco-enzyme– Usually a metal or vitamin

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

Feedback Regulation• Negative feedbackNegative feedback –

pathway is inhibited by accumulation of final product

• Positive feedbackPositive feedback – a regulatory molecule stimulates the activity of the enzyme, usually between 2 pathways ADP levels cause the

activation of the glycolysis pathway to make more ATP

Allostery• Conformational coupling of 2 widely separated

binding sites must be responsible for regulation – active site recognizes substrate and 2nd site recognizes the regulatory molecule

• Protein regulated this way undergoes allosteric transition or a conformational change

• Protein regulated in this manner is an allosteric protein

Phosphorylation

• Some proteins are regulated by the addition of a PO4 group that allows for the attraction of + charged side chains causing a conformation change

• Reversible protein phosphorylations regulate many eukaryotic cell functions turning things on and off

• Protein kinaseskinases add the PO4 and protein phosphatasephosphatase remove them

Phosphorylation/Dephosphorylation

• Kinases capable of putting the PO4 on 3 different amino acid residues– Have a –OH group on R

group• Serine• Threonine• Tyrosine

• Phosphatases that remove the PO4 may be specific for 1 or 2 reactions or many be non-specific

GTP-Binding Proteins (GTPases)• GTP does not release its PO4

group but rather the guanine part binds tightly to the protein and the protein is active

• Hydrolysis of the GTP to GDP (by the protein itself) and now the protein is inactive

• Also a family of proteins usually involved in cell signaling switching proteins on and off

Molecular Switches

Motor Proteins• Proteins can move in the cell,

say up and down a DNA strand but with very little uniformity– Adding ligands to change the

conformation is not enough to regulate this process

• The hydrolysis of ATP can direct the the movement as well as make it unidirectional– The motor proteins that move

things along the actin filaments or myosin

Protein Machines

• Complexes of 10 or more proteins that work together such as DNA replication, RNA or protein synthesis, trans-membrane signaling etc.

• Usually driven by ATP or GTP hydrolysis

• See video clip on CD in book

Functions of Globular 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

Protein Interaction with Other Molecules• Reversible, transient process of chemical equilibrium:

A + B AB

• A molecule that binds to a protein is called a ligand– Typically a small molecule

• A region in the protein where the ligand binds is called the binding site

• Ligand binds via same noncovalent forces that dictate protein structure (see Chapter 4)

– Allows the interactions to be transient

Oxygen Binding Curves

EOC Problem 6 gets you further into cooperativity in oxygen binding.Knowing this will help in Class.

Hemoglobin Binding Curve

Bohr Effect

• Hemoglobin's affinity for oxygen is decreased in the presence of carbon dioxide and at lower pH.

• Carbon dioxide reacts with water to give bicarbonate, carbonic acid free protons via the reaction:

CO2 + H2O ---> H2CO3 ---> H+ + HCO3-

• Protons bind at various places along the protein and carbon dioxide binds at the alpha-amino group forming carbamate.

• This causes a conformational change in the protein and facilitates the release of oxygen.

Bohr Effect

• Blood with high carbon dioxide levels is also lower in pH (more acidic). (recall the equilibrium)

• Conversely, when the carbon dioxide levels in the blood decrease (i.e. around the lungs), carbon dioxide is released, increasing the oxygen affinity of the protein.

Bohr Effect Summary

• High CO2 in tissues • Higher H+• Lower pH• Affinity for O2

decreases• O2 released to tissues• T state favored

• Low CO2 in lungs • Lower H+• Higher pH• Affinity for O2

increases• O2 binds hemoglobin• R state favored

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Disorder?

Amyloid diseases

Disease Protein/peptide Aggregate

Alzheimer’s disease A Senile plaq

Primary systemic amyloidosis Ig light chain

Senile systemic amyloidosis Transthyretin

Diabetes type II Amylin

Hemodialysis-associated amyloidosis 2-microglobulin

Familial systemic amyloidosis Lysozyme mutant

Huntingon’s disease Huntingtin Huntingtin inclusion

Parkinson’s disease -synuclein Lewy body

CJD, other prion diseases PrPSc Prion aggregate

Taupathies, Pick disease, FTDP-17 Tau protein PHF, Pick-body

1) Protein (AL, ATTR, ALys)2) Cause (spontaneous, mutation,

induced)3) Mechanism (loss or gain of

function)

Amyloid diseases: modern classification

Amyloids are insoluble fibrous protein aggregates sharing specific structural traits. They are insoluble and arise from at least 18 inappropriately folded versions of proteins and polypeptides present naturally in the body

protein misfolding diseases

AD plaque Neurofibrillary tangle (PHF)

Alzheimer’s disease

Amyloid precursor protein (APP)

(TACE, ADAM10)

(PSEN)

• Stanley B. Prusiner coined the term proin from Proteinaceous infective particle

and changed to prion to sound it rhythmic.

• Prion diseases were caused by misfolded proteins.

• Elucidated the gene and mechanism by which wild type protein

bring about the

clinical disease.

PRION DISEASES

• Kuru

• Fatal Familial Insomnia (FFI)

• Creutzfeldt-Jakob disease (CJD)

• Scrapie

• Bovine Spongiform Encephalopathy (BSE)

• Chronic Wasting Disease (CWD)

Prion DiseasesPrion DiseasesHumanHuman AnimalAnimal

Classification of prion diseasesClassification of prion diseases• Infectious/ExogenousInfectious/Exogenous

– e.g., Kuru, BSE (mad cow disease), Scrapie– Spread by

• Consumption of infected material.• Transfusion.

• SporadicSporadic

• Familial/HereditaryFamilial/Hereditary– Due to autosomal dominant mutation of PrP.

Differences between cellular and scrapie proteinsDifferences between cellular and scrapie proteinsPrPPrPCC PrPPrPSCSC

SolubilitySoluble Non soluble

Structure Alpha-helical Beta-sheeted

Multimerisation state Monomeric Multimeric

InfectivityNon infectious Infectious

Susceptibility to Proteinase KSusceptible Resistant