© 2003 by default!slide 1 protein sorting, transport and modification part 2 m. saifur rohman, md,...

© 2003 By Default!Slide 1

Protein Sorting, Protein Sorting, Transport and Transport and modificationmodification

part 2part 2

M. Saifur Rohman, MD, PhD, FIHAM. Saifur Rohman, MD, PhD, FIHA


Protein function

1. General features of protein 1. General features of protein functioning;functioning;2. Reversible binding of proteins to 2. Reversible binding of proteins to ligands: ligands: myoglobin and myoglobin and hemoglobin hemoglobin

((for material transport and storagefor material transport and storage););3. Complementary interactions between 3. Complementary interactions between proteins and ligands: immune proteins proteins and ligands: immune proteins ((for defensefor defense););4. Protein-protein interactions: actin and 4. Protein-protein interactions: actin and

myosin (myosin (for motionfor motion).).


Proteins are extremely versatile in structure and function

Complexity and variety in structure Complexity and variety in structure allow proteins to carry out an enormous allow proteins to carry out an enormous arrays of specialized biological tasks:arrays of specialized biological tasks: E.g.,E.g.,– Catalyzing virtually all metabolic reactions;Catalyzing virtually all metabolic reactions;– Enable DNA to carry and duplicate the Enable DNA to carry and duplicate the

genetic information.genetic information.– Play essential roles in building, defending, Play essential roles in building, defending,

and moving the living organisms.and moving the living organisms.

© 2003 By Default!Slide 4Proteins acts in

all biological processes.

proteins

Motion(myosin, actin, etc)

Material transport(hemoglobin, transferrin)

Metabolism(enzymes)

Defense(antibodies, toxins)

Mechanical support(collagen) Replication and

repairing of genetic

information (DNA and RNA polymerases)


Functions of ProteinsFunctions of Proteins


Protein FunctionProtein Function

Very specific biological functionVery specific biological function Varies based on structureVaries based on structure

– Fibrous proteinsFibrous proteins– EnzymesEnzymes– Transport across membranesTransport across membranes

Common theme: how proteins bind to interact with Common theme: how proteins bind to interact with other moleculesother molecules– Involves reversible binding with ligands (or substrates) in Involves reversible binding with ligands (or substrates) in

binding site (or active site)binding site (or active site)– Can have multiple binding sites in one proteinCan have multiple binding sites in one protein– Binding may involve change in conformation (induced fit)Binding may involve change in conformation (induced fit)– Induced fit may cause change in other parts of the protein Induced fit may cause change in other parts of the protein

(e.g. other subunits)(e.g. other subunits)– Interactions between ligands and proteins are regulatedInteractions between ligands and proteins are regulated


Correlation Between Structure & Function

•Homologous proteins

• Conserved sequence, similar structure and function

• Example: cytochrome c

•Similar function, different sequences

• Conserved and variable regions

• Example: dehydrogenases, kinases

•Similar structure, different function

• Example: thioredoxin


Homology Modeling (HM)

Advancement in identification, isolation and sequencing Advancement in identification, isolation and sequencing

of genes – possible to infer sequences of proteins.of genes – possible to infer sequences of proteins.

3D-struc determination – time consuming3D-struc determination – time consuming

Current methods – X-ray diffraction and NMRCurrent methods – X-ray diffraction and NMR

HM helps in generating fairly reasonable models of HM helps in generating fairly reasonable models of

proteinsproteins

Comparative modeling/Knowledge-based modelingComparative modeling/Knowledge-based modeling


What is Homology?

Having a common evolutionary originHaving a common evolutionary origin

Qualitative description of relationships between Qualitative description of relationships between

2 or more things2 or more things

A set of proteins which are homologous – 3D A set of proteins which are homologous – 3D

structure more conserved than their primary structure more conserved than their primary

structurestructure

So, HM involves developing models of unknown So, HM involves developing models of unknown

from homologous proteins.from homologous proteins.


Steps in Homology Modeling

Identify homologous proteins – check extent of similarity Identify homologous proteins – check extent of similarity

b/w them and unknownb/w them and unknown

Align sequencesAlign sequences

Identify structurally conserved regions (SCR) and Identify structurally conserved regions (SCR) and

variable regions (VR)variable regions (VR)

Generate coordinates for core residues of unknownGenerate coordinates for core residues of unknown

Generate conformations for the loops in the unknownGenerate conformations for the loops in the unknown

Build side-chain conformationsBuild side-chain conformations

Refine and evaluate the unknown structureRefine and evaluate the unknown structure


Identifying Homologues

Apply sequence search methodsApply sequence search methods

Identify proteins with seq. similarity to unknown and for Identify proteins with seq. similarity to unknown and for

which 3D struc are availablewhich 3D struc are available

Assume these proteins are homologous and develop a Assume these proteins are homologous and develop a

modelmodel

Ideally many homologues might be present, but possible Ideally many homologues might be present, but possible

to predict even with one homologue to predict even with one homologue


Aligning Sequences

Align sequence of the unknown with its Align sequence of the unknown with its

homologueshomologues

Factors to considerFactors to consider– Algorithm to use for seq. alignmentAlgorithm to use for seq. alignment

• Needleman and Wunsch, Smith and Waterman FASTA, Needleman and Wunsch, Smith and Waterman FASTA,

BLASTPBLASTP

– Scoring method to applyScoring method to apply• PAM, BLOSUMPAM, BLOSUM

– If assigning gap penalties, how to assign themIf assigning gap penalties, how to assign them


Proteins function via interaction

The specialized functions of proteins The specialized functions of proteins can almost all be understood in terms can almost all be understood in terms of how proteins reversibly bind to and of how proteins reversibly bind to and interact with other components interact with other components (called (called ligandsligands) in the living systems.) in the living systems.


Hemoglobin transports O2 and CO2

The red blood cells(each

containing about 300

million HBs)

The hemegroup reversibly

binds O2

Hb: A protein that you can “see” with your naked eyes!

With a structure


Vertebrates use antibodies to defend (i.e., to neutralize antigens)

AntigenAntibody

(IgG: with aH2L2 structure)

The antigenbinding site of an antibody


Muscle contraction depends on the reversible interaction between myosin and

actin

Actin filament

MyosinATP

hydrolysis


Luciferase allows firefly to emit light.

Enzyme catalysis

© 2003 By Default!Slide 18 A few general principles are usually followed for proteins to

function

The interactions between proteins and ligands The interactions between proteins and ligands are usually are usually transienttransient (i.e., non-covalent and (i.e., non-covalent and reversible).reversible).

The interface between the binding site on a The interface between the binding site on a protein and its ligand is protein and its ligand is complementarycomplementary in in structure, making such interactions highly structure, making such interactions highly specificspecific..

The structure of proteins are usually The structure of proteins are usually flexibleflexible and and dynamicdynamic..

Such interactions can be Such interactions can be regulatedregulated..

© 2003 By Default!Slide 19Unveiling the function of a particular protein is usually very difficult

Unlike structure determination, no universal Unlike structure determination, no universal methods are available to reveal the physiological methods are available to reveal the physiological function of the large number of proteins encoded function of the large number of proteins encoded by the genomes.by the genomes.

The The in vitroin vitro properties of a protein is often properties of a protein is often initially investigated before initially investigated before in vivoin vivo functions functions are are studied.studied.

Genomic sequencing revealed a large number of Genomic sequencing revealed a large number of putative proteinsputative proteins whose functions are waiting to whose functions are waiting to be revealed (be revealed (after their actual presence inside after their actual presence inside the cells is confirmedthe cells is confirmed).).


Understanding the function and structural

basis of hemoglobin and myoglobin:

Reversible ligand binding;Regulation of protein

function;Evolution of protein function.

Paradigms of Protein Structure and Function


Hemoglobin

transports O2 and CO2

in mammals.Myoglobin

transports and stores O2 inmuscle tissues


Free iron or heme would not be able to transport O2

Free iron would promote the formation of Free iron would promote the formation of highly active oxygen species (e.g., hydroxyl highly active oxygen species (e.g., hydroxyl radicals).radicals).

One OOne O22 would react with two free heme would react with two free heme molecules, resulting in the irreversible molecules, resulting in the irreversible conversion of Feconversion of Fe2+2+ to Fe to Fe3+3+. .

The Mb and Hb polypeptides cradle the heme The Mb and Hb polypeptides cradle the heme Group (Group (also protects the heme iron atom from also protects the heme iron atom from oxidation, and provides a pocket into which the O2 oxidation, and provides a pocket into which the O2 can fitcan fit). ).


Understanding the structure and function of antibodies

(immunoglobulins)

Proteins found in the blood of vertebrates, bind such foreign invaders (antigens) as bacteria, viruses or large molecules, and target them for destruction.

KKdd=10=10-10-10 M M


The determined structures of antibody-antigen complexes reveal the binding

nature

The antigen binding sites are indeed made of the The antigen binding sites are indeed made of the hypervariable regions (CDRs) of both the H and L hypervariable regions (CDRs) of both the H and L chains (chains (as was hypothesizedas was hypothesized).).

Conformational changes in the antibody and/or the Conformational changes in the antibody and/or the antigen occur, allowing a complementary tight antigen occur, allowing a complementary tight binding between a specific antigen and its specific binding between a specific antigen and its specific antibody.antibody.

Noncovalent interactions are used for the Noncovalent interactions are used for the antibody-antigen interaction.antibody-antigen interaction.

The The KKdd for antibody-antigen interaction can be as for antibody-antigen interaction can be as low as 10low as 10-10-10 M. M.


In this view, the HV regions of the Fab have been deleted: there is no contact between antibody and antigen.

This ribbon structure shows the antibody's HV (purple) region of the Fab, and their interaction with an epitope of the antigen.

Fab Antigen

HV

Antigen


Induced fit conformational changes for the antibody-

antigen interaction.

Induced fit


The antibody-antigen interaction is the basis for a variety of important analytical

techniques

Affinity chromatographyAffinity chromatography: one-step protein : one-step protein purification using a specific antibody that is purification using a specific antibody that is covalently attached to a resin.covalently attached to a resin.

Immunoblot assayImmunoblot assay:: including Western blotting and including Western blotting and Enzyme-linked immunosorbent assay (ELISA); Enzyme-linked immunosorbent assay (ELISA); qualitative and quantitative detection of a qualitative and quantitative detection of a specific specific antigen present in a mixtureantigen present in a mixture, which could be a , which could be a solution, a gel, or a living cell, using its solution, a gel, or a living cell, using its specific specific antibody that is labeledantibody that is labeled (e.g., being linked to an (e.g., being linked to an enzyme).enzyme).


(antibody, receptor etc) Specific antigen

Nonspecific proteins

Salt solution or other agent

Only Specific antigens are retained on the column


The principle and procedure of immunoblot assays

Secondaryantibody(labeled)

Primary antibody(antigen specific)

Colorlesssubstrate

Colored product(for easy detection)

Protein mixtures are asborbed to aninert surface

Horseradishperoxidase


Western blotting to detect a specific antigen protein in a protein mixtureusing a specific

antibody.Gel-separated antigen

proteins are transferred onto a nitrocellulosemembrane before being probed with

antibodies.


Understanding the roles of myosin and

actin in the contraction of

skeletal musclesA paradigm for how proteins A paradigm for how proteins

translate chemical energy into motion.translate chemical energy into motion.


Protein-based molecular motors allow organisms to “move”at various levels

Many things are moving in living organisms, e.g.:Many things are moving in living organisms, e.g.:– Muscle contraction in vertebrates (Muscle contraction in vertebrates (myosin and actinmyosin and actin));;– Migration of organelles along microtubules and Migration of organelles along microtubules and

chromosome separation in dividing cells(chromosome separation in dividing cells(kinesins and kinesins and dyneinsdyneins););

– Rotation of bacterial flagella (Rotation of bacterial flagella (a complex rotational motor a complex rotational motor proteinsproteins););

– DNA metabolism (DNA metabolism (helicases and polymeraseshelicases and polymerases).). Via cyclic conformational changes of proteins, Via cyclic conformational changes of proteins,

consuming chemical energy supplied by ATP.consuming chemical energy supplied by ATP. Achieving exceptionally high levels of spatial and Achieving exceptionally high levels of spatial and

temporal organization.temporal organization.


Myosin structure: EM photoand schematic drawing


Myosin can be cleaved into smaller fragments by proteases


Structure of the S1 fragment of

myosin

Actin binding

site

Nucleotide binding site

Light chains

The motor domain


Purified myosin forms bipolar aggregates: with the tail stacking one on another.

(Each is made of several hundred myosin molecules)


Purified actin (G-actin) associates to form long filaments or F-actin.

G-actin


Troponin-C

1) F-actin - a polymer of many G-actin monomers

F-Actin[Tropomyosin

Troponin-I

Troponin-T

G-Actin monomer

2) Tropomyosin - lies in the groove between F-actin strands and blocks myosin binding sites on actin.

3) Troponins (Tn): Tn-I binds to actin & inhibits actomyosin interaction: Tn-T binds to tropomyosin;Tn-C binds to Ca2+.

A complex of multiple interacting proteins on F-actin regulate myosin-

actin interaction.


EM photos of a relaxed (top) and contracted (bottom) muscles

Structure of skeletal muscle

Z diskSarcomere （肌节） :

The functional unit of myofibril

Actin and myosin make up more than80% of the protein mass of muscle!

(each is a single multinucleated cell)


Thick filament Thin filament

Cross-bridge (revealed by EM, 1958)

Arrangement of the thin (actin) and thick (myosin) filaments

The sliding model of muscle contraction: thick and thin filaments slide over each other

Each thick filament is surrounded by 6 thin filaments


Proteins other than myosin and actin are also found in muscle

They form the Z disk (e.g., They form the Z disk (e.g., -actinin, desmin, and -actinin, desmin, and vimentin) and the M disk (e.g., paramyosin, C-vimentin) and the M disk (e.g., paramyosin, C-protein, and M-protein); protein, and M-protein); organize the arrays of organize the arrays of thick and thin filaments (e.g., titin and nebulin);thick and thin filaments (e.g., titin and nebulin); regulate myosin-actin interaction (e.g., regulate myosin-actin interaction (e.g., tropomyosin and troponin)tropomyosin and troponin)..

Titin is the largest polypeptide (~ 27,000 residues) Titin is the largest polypeptide (~ 27,000 residues) so far found in nature, it extends from the Z disk to so far found in nature, it extends from the Z disk to the M disk, linking the thick filaments to the Z disk.the M disk, linking the thick filaments to the Z disk.

Nebulin, having ~7,000 residues, is thought to be Nebulin, having ~7,000 residues, is thought to be structures as an a helix spending the length of the structures as an a helix spending the length of the thin filament.thin filament.


Titin: Extends from the Z disk to the M disk.

Titin is the largest polypeptide so far found in nature.

Ig domain


Detailed studies of the myosin-actin interaction led to a molecular

understanding of the muscle contraction process

Myosin with bound ATP has a low affinity for actin Myosin with bound ATP has a low affinity for actin (ATP dissociates the actomyosin interaction), but the (ATP dissociates the actomyosin interaction), but the “primed” head (with bound ADP and Pi) binds readily.“primed” head (with bound ADP and Pi) binds readily.

The release of Pi from myosin provokes a major The release of Pi from myosin provokes a major rotation, or “power stroke,” of the lever arm. rotation, or “power stroke,” of the lever arm.

Dissociation of ADP from myosin generate a change Dissociation of ADP from myosin generate a change of conformation for the myosin head. of conformation for the myosin head.

The mechanism of transduction of chemical energy, The mechanism of transduction of chemical energy, derived from ATP hydrolysis, into mechanical work derived from ATP hydrolysis, into mechanical work was detailed in the “power stoke” model (1971).was detailed in the “power stoke” model (1971).


Step 1: Release of Ca2+---allows binding of myosin head to the actin filament.

Step 1Ca2+

ADP+Pi

Myosin filamentHigh actin affinity

Resting muscle

Binding of myosin head to actin filament

ADP+Pi

*

Crossbridge[Ca2+]i

Rest ~10-8 MStim ~10-5

M

Actin filament


ADP+Pi

*

ADP+Pi

Binding of myosin head to actin

filament

Pi

ADP

Power Stroke

*Tilting of myosin head

results in power stroke

Step 2

Power StrokePower StrokePower StrokePower Stroke

Ca2+Step 2: Release of ATP hydrolysis products causes a change in myosin head conformation that results in the power stroke and generation of increased tension.

Moving about 2-10 nm for each cycle.

Resting muscle

Step 1


*

ADP+Pi

ADP+Pi

Pi

ADP

*

ATP

Low actin affinity

*

Step 3 ATP

Ca2+

Step 3: Binding of ATP to myosin head stimulates the release of myosin from actin filament. Step 1

Step 2


High actin affinity

ADP+Pi

Pi

ADP

*

Step 4

ATP

Step 4: Rapid hydrolysis of ATP causes change in myosin head conformation to return to high actin affinity state.

Low actin affinity

*

ATP

*

ADP+Pi

Ca2+

Step 1

Step 2

Step 3


Cycle stops here in relaxed living muscle

(due to removal of myoplasmic calcium)

Cycle stops here in absence of ATP (rigor mortis)

Ca2+

*High actin affinity

ADP+Pi

ADP+Pi

Pi

ADP

*

Low actin affinity

*

ATP

ATP

Resting muscle

Rigor Complex


Muscle contraction depends on the reversible interaction between myosin and actin, as well as the ATPase activity of the

Myosin

Actin filament

Myosin

ATP hydrolysis

Conformationalchanges in the myosin head that are coupled to stages in the ATP hydrolytic cycle cause myosin to successively dissociate from oneactin subunit, then associate with anotherfarther along the actin filament.


The molecular understanding of

contraction of the skeletal muscle provides a

paradigm for understanding the molecular process of

other motions in the living organisms


Summary

Proteins are extremely versatile in structure and Proteins are extremely versatile in structure and function.function.

Proteins function via interaction.Proteins function via interaction. Myoglobin and hemoglobin both reversibly binds Myoglobin and hemoglobin both reversibly binds

OO2 2 through the heme groups, each with binding through the heme groups, each with binding and regulatory properties beautifully satisfying and regulatory properties beautifully satisfying requirements for their physiological roles. requirements for their physiological roles.

Antibodies are vertebrate proteins evolved for Antibodies are vertebrate proteins evolved for removing potentially harmful foreign antigens of removing potentially harmful foreign antigens of tremendous variation in structure.tremendous variation in structure.

Myosin and actin has evolved as a pair of proteins Myosin and actin has evolved as a pair of proteins that act in generating cellular motions inside the that act in generating cellular motions inside the cells or for the cells.cells or for the cells.

© 2003 by default!slide 1 protein sorting, transport and modification part 2 m. saifur rohman, md,...

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