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Proteins - are the working molecules of a cell which carry out the programof activities encoded by genes

Enzymes- substances capable of catalyzing an incredible range ofintracellular and extracellular chemical reactions with speedand specificity

Proteome- refers to the entire protein complement of an organism

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- provide structural rigidity to the cell

- control the flow of materials across

cellular membranes

- act as sensors and switches to control

protein activity and gene function

- transmit external signals to the cellinterior (includes cell surface receptors)

- cause motion

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Key Concepts:

Only when a protein is in its correct three-dimensional structure, orconformation, is itable to function efficiently.

Function is derived from three-dimensional structure, and three-dimensional structure is

specified by amino acid sequence.

Primary Structure of Proteins (Linear)

Structure of a Tripeptide

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Primary structure of a protein is simply the linear arrangement, or sequence, ofthe amino acid residues that compose it.

Peptide- a short chain of amino acids linked by peptide bonds and havinga defined sequence.

Polypeptide- longer chains of amino acids

Secondary Structure of Proteins

- Helix

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Tertiary Structure of Proteins

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(a) Two helices connected by a short loop in aspecific conformation constitute a helix-loop-helix motif. This motif exists in manycalcium-binding and DNA-bindingregulatory proteins.

(b) The zinc-finger motif is present in manyDNA-binding proteins that help regulatetranscription.

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o The simplest way to represent three-dimensional structure is to trace the course of the

backbone atoms with a solid line (fig. A) while the most complex model shows every

atom(fig. B).

o The solvent-accessible surface model (fig. D) conveys information about the protein

surface where other molecules bind. On this water-accessible surface, regions having a

common chemical character (hydrophobicity or hydrophilicity) and electrical character

(basic or acidic) can be mapped.

Quaternary Structure of Proteins

(Multimeric)

- consist of three or more polypeptides or subunits

- describes the number (stoichiometry) and relativepositions of the subunits in multimeric proteins

- (at the right) Hemagglutinin, a quaternary structure

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Macromolecular Assemblies

o an example is the capsid that encases the viral genome and bundles of cytoskeletalfilaments that support and give shape to the plasma membrane

o other macromolecular assemblies act as molecular machines, carrying out the most

complex cellular processes by integrating individual functions into one coordinated process

mRNA Transcription Machineries

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Protein folding is needed to generate the structure of the final protein.

Incorrectly folded proteins usually lack biological activity and, in some cases, mayactually be associated with disease.

To correct misfoldings, the cell has error-checking processes that eliminateincorrectly synthesized or folded proteins.

Any polypeptide chain containing n residues could fold into 8n conformations.

native state- the most stably folded form of the molecule

What guides proteins to their native folded state? protein folding is a self-directed process sufficient information must be contained in the proteins primary sequence to direct

correct folding

In vitro studies showed that: Thermal energy from heat extremes of pH Chemicals

These factors disrupt the weak noncovalent interactions that stabilize the native

conformation of a protein.

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Although protein folding occurs in vitro, only a minority of unfolded molecules undergo complete

folding into the native conformation within a few minutes.

Chaperones- a class of proteins found in all organisms from bacteria to humans

- are located in every cellular compartment

- function in the general protein-folding mechanism of cells

Two general families of chaperones are recognized:

Molecular chaperones - bind and stabilize unfolded or partly folded proteins

Chaperonins- directly facilitate the folding of proteins

Chaperone- mediated Protein Folding

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Chaperones function is to assist the assembly process,they do not convey additional information necessary for folding into

the correct conformation.

The folded conformation is determined by the amino acid sequence.

- function by stabilizing unfolded or partially foldedpolypeptides that are intermediates along the pathway leading to

the final correctly folded state.

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Chaperones stabilize unfolded proteins which prevents the

accumulation of incorrectly folded proteins. As you know protein misfolding

could have very bad consequences.

For example, misfolded proteins can aggregate to form insoluble

fibers called amyloid fibers. These fibers accumulate in the extracellular

spaces and within cells and are characteristic of several

neurodegenerative disease such as Parkinsons and Alzheimers disease.

Two types of chaperone proteins:

1) heat shock proteins and

2) chaperonins Found in the cytosol and sub

cellular organelles of eukaryotic

cells

* HSP (heat shock protein) - stabilize and facilitate the refolding of

proteins that have been partially denatured as a result of exposure to

elevated temperatures.

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They bind short hydrophobic regions of unfolded polypeptides. This maintains the

polypeptide chain in an unfolded conformation and prevents aggregation.

The E.coliHsp70 which include proteins called Hsp70coded by dnaK gene, and

Hsp40 coded by dnaJ gene and the GrpE.

The chaperons bind to hydrophobic regions of the proteins, including proteinsthat still are being translated. They prevent aggregation by holding the protein in an

open confirmation until it is completely synthesized and ready to fold.

They also are involved in other processes that require shielding of hydrophobic

regions.

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*Chaperonins function to protect unfolded polypeptides

from proteolysis in the cytoplasm.

Chaperonins consist of multiple protein subunits

arranged in two stacked rings to form a double chambered

structure. This allows protein folding to occur without

aggregation of unfolded segments.

The main version of which is E. coli is GroEL/GroEs

complex. The complex is a multisubunit structure that looks

like a hollowed-out bullet. Protein enters unfolded and existsfolded.

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Chaperonin-Mediated Protein Folding

Mechanisms:1. Partly folded or misfolded polypeptide is inserted into the cavity of GroEL.

2. The protein then binds to the inner wall and folds into its native

conformation.

3. In an ATP-dependent step, GroEL undergoes a conformational change

and releases the folded protein, a process assisted by GroES.

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Acetylation - the addition of an acetyl group (CH3CO) to the amino group of the N-terminal residue

Phosphorylation - addition and removal of phosphate groups from serine, threonine, or tyrosine residues

Glycosylation - the attachment of linear and branched carbohydrate chains

Hydroxylation -introduction of one or more hydroxyl groups (-OH) into a compound (or radical) Methylation -the attachment or substitution of a methyl group on various substrates

Carboxylation -the addition of carbon dioxide or bicarbonate to form a carboxyl group

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1. Enzyme 1 (E1) is activated by attachment of a

ubiquitin (Ub) molecule.

2. It then transfers this Ub molecule to E2.

3. Ubiquitin ligase (E3) transfers the bound Ub

molecule on E2 to the side-chain NH2 of a

lysine residue in a target protein.

4. Additional Ub molecules are added to the target

protein by repeating steps, forming apolyubiquitin chain that directs the tagged

protein to a proteasome.

5. Within this large complex, the protein is cleaved

into numerous small peptide fragments.

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-In eukaryotic cells there are 2 major pathways thatmediate protein degradation:

1) ubiquitin-proteasome

2)lysosomal proteolysis pathway whichinvolves the uptake and digestion of proteins by thelysosomes.

-Many rapidly degraded proteins function as regulatorymolecules such as transcription factors, particularly thosethat are involved in mediating cell proliferation and cellsurvival.

-This allows for their levels to change veryrapidly in response to external stimuli.

-Other proteins are rapidly degraded in response tospecific signals. Also, faulty or damaged proteins are

recognized and rapidly degraded within cells, therebyeliminating the consequences of mistakes made duringprotein synthesis.

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Digestive Proteases Endoproteases- attack selected peptide bonds within a polypeptide chain.

The principal endoproteases are pepsin, which

preferentially cleaves the backbone adjacent to

phenylalanine and leucine residues, and trypsin and

chymotrypsin, which cleave the backbone adjacent to

basic and aromatic residues.

Exopeptidases- sequentially remove residues from the N-terminus

(aminopeptidases) or C-terminus (carboxypeptidases) of

a protein.

Peptidases - split oligopeptides containing as many as about 20

amino acids into di- and tripeptides and individual amino

acids.

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Two Properties of Protein-Ligand Interaction:

1. Specificity - the ability of a protein to bind one molecule in preference to other

molecules

2. Affinity -the strength of binding

Both the specificity and the affinity of a protein for a ligand depend on the structure of the

ligand-binding site.

For high-affinity and highly specific interactions to take place, there must be molecularcomplementarity.

An example is the antibody-antigen complex:

The hand-in-glove fit between an antibody and an

epitope on its antigenin this case, chicken egg-

white lysozyme. Regions where the two moleculesmake contact are shown as surfaces.

The antibody contacts the antigen with residues

from all its complementarity-determining regions

(CDRs).

In this view, the complementarity of the antigen

and antibody is especially apparent where

fingers extending from the antigen surface are

opposed to clefts in the antibody surface.

E

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Enzymes Catalysts of chemical reactions in cells.

Increase the reaction rates by lowering the activation energy of chemical reactions

Two Striking Properties of Enzymes:Catalytic power- causes the rates of enzymatically catalyzed reactions to be 1061012 times that of

their corresponding uncatalyzed reactions under similar conditions.

Specificity - ability to act selectively on one substrate or a small number of chemically similarsubstrates

Effect of a Catalyst on the Activation Energy of a Chemical Reaction

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Evolution of Multifunctional Enzyme

(a) When the enzymes are free in solution or even constrained within the same cellular compartment, theintermediates in the reaction sequence must diffuse from one enzyme to the next, an inherently slowprocess.

(b) Diffusion is greatly reduced or eliminated when the enzymes associate into multisubunit complexes.

(c) The closest integration of different catalytic activities occurs when the enzymes are fused at the genetic level,becoming domains in a single protein.

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Motor Proteins - specialized enzymes used by the cells to generate the forcesnecessary for many cellular movements- generate either linear or rotary motion

Three general properties of motor proteins:

1. The ability to transduce a source of energy, either ATP or an ion gradient, into linear or rotary

movement2. The ability to bind and translocate along a cytoskeletal filament, nucleic acid strand, or proteincomplex

3. Net movement in a given direction

Linear and Rotary Motor Proteins

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Allostery- refers to any change in a proteins tertiary or quaternary structure or bothinduced by the binding of a ligand, which may be an activator, inhibitor,substrate, or all three.

Ligand-induced Activation of Protein Kinase A (PKA) Switching Mediated by Ca2+/Calmodulin

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Regulation of Protein Activity by Kinase/Phosphatase Switch

Phosphorylation- addition and removal of phosphate groups from serine, threonine,

or tyrosine residues

Protein kinases catalyze phosphorylation while phosphatases catalyzedephosphorylation.

All classes of proteinsincluding structural proteins, enzymes, membrane channels, andsignaling moleculesare regulated by kinase/phosphatase switches.

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Purifying Techniques1. Centrifugation

Principle:

Two particles in suspension (cells, organelles, or molecules) with

different masses or densities will settle to the bottom of a tube at

different rates.

Centrifugation is used for two basic purposes:

(1) as a preparative technique to separate one type of material from others(2) as an analytical technique to measure physical properties of macromolecules

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DIFFERENTIAL CENTRIFUGATION RATE-ZONAL CENTRIFUGATION

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- a technique for separating molecules in a mixture under the influence of an appliedelectric field.

Principles:

When a mixture of proteins is applied to a gel and an electric current is applied,smaller proteins migrate faster through the gel than do larger proteins.

The rate at which a protein moves through a gel is influenced by the gels poresize and the strength of the electric field.

By suitable adjustment of these parameters, proteins of widely varying sizescan be separated.

Types of Electrophoresis

1. SDS-Polyacrylamide Gel Electrophoresis

2. Two- Dimensional Gel Electrophoresis

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SDS- POLYACRYLAMIDE GELELECTROPHORESIS

TWO-DIMENSIONAL GELELECTROPHORESIS

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Western Blotting (Immunoblotting) Step 1 : After a protein mixture has been electrophoresed through an SDS gel, the separated bands are transferred

(blotted) from the gel onto a porous membrane.

Step 2 : The membrane is f looded with a solution of antibody (Ab1) specific for the desired protein. Only the bandcontaining this protein binds the antibody, forming a layer of antibody molecules . After sufficient time for binding, themembrane is washed to remove unbound Ab1.

Step 3 : The membrane is incubated with a second antibody (Ab2) that binds to the bound Ab1. This second antibodyis covalently linked to alkaline phosphatase, which catalyzes a chromogenic reaction.

Step 4: Finally, the substrate is added and a deep purple precipitate forms, marking the band containing the desiredprotein.

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a) When a narrow beam of x-rays strikes

a crystal, part of it passes straight

through and the rest is scattered

(diffracted) in various directions. The

intensity of the diffracted waves is

recorded on an x-ray film or with asolid-state electronic detector.

b) X-ray diffraction pattern for a

topoisomerase crystal collected on a

solid-state detector. From complexanalyses of patterns like this one, the

location of every atom in a protein can

be determined.

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