Mathews et al (2000) Biochemistry (3e) p.164

• MOLECULAR CELL BIOLOGY

• SIXTH EDITION

• MOLECULAR CELL BIOLOGY

• SIXTH EDITION

Copyright 2008 ©W. H. Freeman and Company

• CHAPTER 3

• Protein Structure and Function

• CHAPTER 3

• Protein Structure and Function

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

© 2008 W. H. Freeman and Company

Human signaling

protein keapl

Ribbon diagram

Conformations

Structural proteins Scaffold protein

Transport protein Regulatory protein enzymes

Functional classes:

Structural proteins Transport proteins Regulatory proteins Motor proteins

Different conformation = different function

Proteins are single, unbranched chains of amino acid monomers

There are 20 different amino acids; All amino acids have the same general structure but the side chain (R group) of each is different

A protein’s amino acid sequence determines its three-dimensional structure (conformation)

In turn, a protein’s structure determines the function of that protein

Hierarchical structure of proteins

H₃ N C COO H

R

pK₁ pK₂

+ -

Primary: the linear sequence of amino acids

Secondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet)

Tertiary: the overall, three-

dimensional arrangement of the polypeptide chain

Quaternary: the association of two or more polypeptides into a multi-subunit complex

Four levels of structure determine the shape of proteins

Four levels of protein

hierarchy

1^o 2 ^o 3^o 4^o

一級primary 二級secondary 三級tertiary 四級quaternary

Nelson & Cox (2000) Lehninger Principles of Biochemistry

Amino acid 2 amino acids peptide polypeptide

Peptide bond - linkage

between amino acids is a secondary amide bond Formed by condensation of

the α-carboxyl of one amino acid with the α-amino of

another amino acid (loss of H₂ O molecule)

Primary structure - linear

sequence of amino acids in a polypeptide or protein

The primary structure of a protein is its liner arrangement of amino acid

The backbone of protein (polypeptide)

N-C-C-N-C-C-N-C-C-N-C- N C C

N-terminal C-terminal

Unit (單位)

Peptide bond (胜鍵)

Secondary structure: the α helix Secondary structure are the core

elements of protein architecture

每 3.6 胺基酸繞一圈，每圈 5.4 Å 高

Carbonyl (C=O) 與下游 H-N- 生成 氫鍵

Hydrogen bonds determine water solubility of uncharged molecules

局部負電性

Intra-molecular Inter-molecular

Covalent bond

未共用之電子對

Secondary structure: the beta sheet

Structure of a β turn

1. A short U-shaped beta turn 2. Four residue

3. H-bond stable

4. Proline and glycine present

β turn γ turn

Pro

劇烈轉折 R 在同一側

R 在相對側

三個胺基酸夾一氫鍵 兩個胺基酸夾一氫鍵

Mathews et al (2000) Biochemistry (3e) p.181

Reverse Turns

Reverse Turns: β turn, γ turn

It also related with H-bond

Oil drop model of protein folding

Hydrophobic interact with hydrophobic Hydrophilic interact with hydrophilic

Hydrophobic nonpolar Hydrophilic polar

Uncharged hydrophilic polar side chains are found on both the surface and inner core of protein

04_05_Hydrophobic.jpg

Integral membrane protein

Globular protein

Overall folding of a polypeptide chain yields its tertiary structure

帶狀 溶劑親水表面

球與棒

Different graphical representations of the same protein

Ras

GDP

Different way of depicting the conformation of proteins convey different types of information

Red: negative charge Purple: positive charge

Motifs are regular combination of secondary structures

Motif: particular combinations of secondary structures, it build up the tertiary structure of a protein; super-secondary strucure and 2-5 secondary structure

Hydrophobic interaction

Structural and functional domains are modules of tertiary structure

(a) Tertiary structure (b) Quaternary structure

Domain

Hemagglutinin(流行性感冒表面蛋白質-血細胞凝集素) 3 subunit

Mathews et al (2000) Biochemistry (3e) p.164

α helix β sheet

兩者都由

H-bond組成

all α helices all β sheets helices + sheets

Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.26

Secondary structure produced Tertiary structure

turn

Structural and functional domains are modules of tertiary structure

Various proteins illustrating their modular nature

domain

Epidermal growth factor (EGF) is generated by proteolytic cleavage of a precursor protein.

These proteins also contain other widely distributed domains indicated by shape and color

Tissue plasminogen activator

Two or more polypeptides or subunit → multimeric protein

Quaternary structure: a fourth level of structural organization; it describes the number and relative positions of subunits in multimeric protein.

The highest level of protein structure is the association of protein into macromolecular assemblies.

Quaternary structure

Folding, modification, and degradation of proteins

• A newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein

• All molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule

The mRNA transcription-initiation machinery

Proteins associate into multimeric structures and macromolecular assemblies

Sequence homology suggests functional and evolutionary relationships between proteins

Homology: have a common ancestry are referred to as homologs. It is similarity in their sequence or structure.

Members of protein families have a common evolutionary ancestor

Similarity homology homologs

Different amino acid sequence ----> different conformation - different function

High sequence similarity about >50 % : related structure or function

Family and superfamily

Family protein about >30% amino acid sequence similarity

Folding, modification, and degradation of proteins

The information for protein folding is encoded in the sequence Conformational folding can denature to polypeptides

Planar peptide bonds limit the

shapes into which protein can fold

Information directing a protein’s folding is encoded in its amino acids sequence

Denature or denaturation : disrupt noncolavent interaction

Only colavent bond: disulfide bond need beta-mercaptoethanol

Folding of protein in vivo is promoted by chaperones

SBD

Chaperone-mediate protein folding HSP: heat shock protein

Members of Hsp70 family (homologs)

• DnaK (bacteria)

• Hsp70 (cytosol, mitochondrial of eukaryotic cells)

• BiP (endoplasmic reticulum)

Molecular chaperone

Co-chaperone : Hsp 40/DnaJ

Protein folding from primary to final

Primary structure dictates final structure but most proteins cannot assume final conformation without help.

Chaperones provide this help.

Few proteins can achieve their active conformation unaided. (it must need help)

During stress proteins unfold and need to reassemble. Note that chaperones are also called heat shock proteins (Hsp).

Protein complexes may require help from a chaperone to form; other complexes may require help to be broken down.

Chaperones are necessary

Chaperonin mediated protein folding

GroEL/Hsp60 system (Chaperonin)

Constitutively expressed and increased in response to stress One of the key chaperone systems for most cytosolic proteins

Note that it is also important in protein translocation and degradation GroEL is chaperone (Hsp60)

GroES is regulatory protein Structure

– GroEL 14 x 57 kDa (2 rings of 7) – GroES 7 x 10 kDa

inner surface is hydrophobic interact with hydrophobic region of polypeptides

note that native (folded ) proteins do not bind

GroES binds to GroEL

GroEL: cylindrical (圓柱形) structure ;

two heptameric rings of ~57 kDa subunits (7)

GroES: ;dome-shaped;

heptameric ring of 10 kDa subunits (7)

Two families of molecular chaperone for protein folding:

DnaK/DnaJ/GrpE (or hsp70) family: bind to growing polypeptide chains while they are being synthesized by ribosomes and prevent premature folding (co-translational)

Chaperonin family (GroE chaperonin): assist correct folding at a later stage (post-

translational)

• Molecular chaperons

– Binds to unfolded and partially folded proteins to prevent in proper association of exposed hydrophobic patches ( bind to hydrophobic part)

– Assist folding of larger multidomain proteins

– Heat shock proteins (rate of their syntheses increases with at elevated temperature)

– Hsp70 – monomeric 70 kDa proteins

• Binds to newly synthesised protein peptide emerging from the ribosome

– Hsp90 proteins – involved in the folding of proteins participating in signal transduction (steroid hormon receptors etc).

Chaperonins – large multisubunit proteins

Folding of protein in vivo is promoted by chaperones

Molecular chaperones: bind and stabilize unforlded or partly folded proteins, preventing these proteins from aggreating and being degraded

Chaperonin: directly facilitate the folding of proteins

Have ATP

No ATP

Alternatively folded proteins are implicated in slowly developing diseases Alzheimer’s disease

Insoluble plaques composed of amyloid protein from unknown mechanism of proteolysis of the amyloid precursor protein.

Misfolding protein

α-helix → beta sheet

Beta-amyloid Plaques

Amyloid precursor protein (APP) is the precursor to amyloid plaque.

1. APP sticks through the neuron membrane.

2. Enzymes cut the APP into fragments of protein, including beta-amyloid.

3. Beta-amyloid fragments come together in clumps to form plaques.

Protein folding

Specific and affinity of protein-ligand binding depend on molecular complementarity

Ligand: the molecule to which a protein binds is often called it Specificity: the ability of molecular and molecular interaction Affinity: tightness or strength of binding

Kd: affinity usually use dissociation constant = 1/Keq

Ligand binding site: both specificity and affinity of a protein for a ligand depend on the structure

Keq = --- Kf Kr

A + B C Kf Kr

Kd= ---

P + L C [P][L]

[C]

Dissociation constants of binding reactions reflect the affinity of interacting molecules

Kd: dissociation constants Kd ↑ non-specific

↓ more specific

Kd: dissociation constant of receptor-ligand complex; ↓ complex more good

RL →response

RT: total receptor number

Dissociation Constant (Kd): is the free ligand conc at which 50% of receptor is occupied.

Kd represents affinity of ligand binding to receptor (1 affinity).

Each ligand has its own specific affinity to the receptor. This can be used to define a new drug or confirm a

receptor.

R + L ⇔ RL

[R][L] K off K_D = --- = ---

[RL] K on Given [R_T ] = [R]+[RL]

[RL]/RT= the fraction of receptors that have bond ligand

Derive the following equation:

[RL] 1

--- = --- R_T 1 + K_D /[L]

Specific and affinity of protein-ligand binding depend on molecular complementarity

Molecular complementarity: High affinity and specific interaction to take place, the shape and chemical surface of binding site must be complementary to ligand moleculae Antibody

Antigen

CDR: complementarity-determining regions

Epitope

affinity and specificity

Enzyme are highly efficient and specific catalysts

A reaction will take place spontaneously only if the total G of the products is less than that of reactants.

All chemical reactions→high energy transition state→ rate of reaction is inversely to G → So need enzyme for catalysts

Active site of the enzyme trypsin

Highly efficient and specific catalysts

An enzyme active site binds substrates and carries out catalysis Active site: specific and chemical reaction site

Michaelis-Menten equation:

在足夠的基質濃度下，一定量的酵素所能催化 的最高反應速率，即為其 Vmax 。

要讓一個酵素達致其 Vmax，就要把基質量調至 最高濃度。

Vmax

若酵素的 Km 越低，則表示它要接近 Vmax 所需的基質濃度越 低。

若某一酵素有數種基質，各有不同的 Km，則

Km 越低的基質，表

示它與酵素的親和力越大，催化反應愈容易進行。

Km 與 [S] 一樣是濃度單位 (mM 或 mM)。

Enzyme can

enhance reaction

No enzyme E: enzyme

Substrate

binding in the

active site of

protease

pH dependence of enzyme activity

Enzyme inhibitor

Enzymes in a common pathway are often physically associated with one another

Evolution of multifunctional enzyme

Diffusion → very slow

Complex subunit

Association

Integration of different catalytic activities in a single protein

Enzyme called molecular motors convert energy into motion

Molecular motors (motor protein): generate the forces necessary for many cellular movements, cells depend on specialized

enzymes.

Mechanochemical enzyme

Regulation protein function I: PROTEIN DEGRADATION

Regulation protein fuction II: Noncovalent and covalent modification

Synthesis

degradation

Ubiquitin marks cytosolic proteins for degradation in proteasomes

Degradation of protein

1. Lysosome: primarily toward extracellular protein and aged or defective organelles of the cells. Membrane

organelles.

2. Proteasomes: Ubiquitin

dependent; for intracellular unfolding, aged protein.

1. control native cytosolic protein

2. misfolded in the course of their synthesis in the ER

THE PROTEASOME

UBIQUITIN 76 Amino Acid polypeptide

3 Amino acid differences between yeast and human homologues

C-Terminal Gly residue is activated via an ATP to form a thiol ester Found only in eukaryotic organisms and is not found in either

eubacteria or archaebacteria.

Among eukaryotes, ubiquitin is highly conserved, meaning that the amino acid sequence does not differ much when very

different organisms are compared.

Ub is a heat-stable protein that folds up into a compact globular structure.

Degradation of a Protein Via the Ubiquitin-Proteasome Involves Two Successive Steps

1. Covalent attachment of multiple ubiquitin molecules to a protein substrate.

2. Degradation of the tagged protein by the 26s proteasome.

(ubiquitin is recycled)

Ubiquitination: In general, multiple ubiquitin units are arranged in polyubiquitin chains linked via Lys₄₈ of ubiquitin, targeting the protein for degradation

Ubiquitin Conjugation:

A 3 Step Mechanism

Ubiquitin (Ub) activating enzyme E

High energy thiol ester is

formed between C-terminal Gly of ubiqutin and a Cys in the E

active site (ATP/AMP)

Ubiquitin conjugating enzymes E

Ub is transferred to a Cys of E

forming a new thiol ester

Ubiquitin ligase E

Ub forms isopeptide bond

between C-terminal Gly of Ub

and ε-amino group of Lys on a

target protein

Functional design of proteins

Protein function generally involves conformational changes Proteins are designed to bind a range of molecules (ligands)

– Binding is characterized by two properties: affinity and specificity

Antibodies exhibit precise ligand-binding specificity Enzymes are highly efficient and specific catalysts

– An enzyme’s active site binds substrates and carries out catalysis

affinity and specificity

Mechanisms that regulate protein function

Allosteric transitions

– Release of catalytic subunits, active ⌦ inactive states, cooperative binding of ligands

Phosphorylation ⌦ dephosphorylation Proteolytic activation

Compartmentalization

Regulation protein fuction II: Noncovalent

and covalent modification

Noncovalent binding permit allosteric, or cooperative, regulation of protein

Allostery: other shape, change protein 3 or 4 structure Allosteric protein

Allosteric effector

Allosteric binding site Cooperativity

Factor bind to → protein A site (noncovalent)→ change protein structure → affect other binding site (activity site)

→ allosteric effect ; when factor = protein, also called

allosteric protein; its binding site also celled allosteric

binding site

Allostey: any change in a protein’s 3 or 4 structure or both induced by the binding of a ligand (activator, inhibitor substrate)

結合一個後→讓另一個更容易結合 (postive regulation) 釋放一個後→讓另一個結合力下降→更容易釋放

High affinity

Flexible

異位性調節

Allosteric protein – a protein in which the binding of a ligand to one site affects the binding properties of

another site on the same protein (also called induced fit model). The conformational changes induced by the modulator(s

modulator(s)) interconvert more-active and less-active forms of the protein.

allos --- other

stereos --- solid or shape

Homotropic interaction --- liagnd = modulator Heterotropic interaction --- ligand = modulator

O² --- as both a normal ligand and an activating homotropic modulator for Hb

The modulators for allosteric proteins may be either inhibitors or activators

Allosteric control: either an activator or inhibitor acts on a portion of the enzyme other than the active site to regulate enzyme function.

Allosteric transition between active

and inactive states

Ligand-indced activation of protein kinase Ligand binding can induce allosteric release of

catalytic subunits or transition to a state with different activity

Allosteric release of catalytic subunits

Switch mediated by Ca ²⁺/calmodulin In normal condition:

cytosolic calcium is low 10^-7 M by ER or pump.

ER release calcium to 10-100 fold → sense calmodulin →

conformal change → regulated other

protein or molecule

Ca ²⁺

Cycling of GTPase switch proteins between the active and inactive forms

Regulation of protein activity by

kinase/phosphatase switch

Many proteins undergo chemical modification of amino acids residues

20 amino acid → chemical modification →100 up Acetylation: about 80% chemical modification Phosphorylation: serine, threonine, tyrosine Glycosylation

hydroxylation Methylation carboxylation

Mainly in actin

Mainly in prothrombin, an essential blood clotting factor

Mainly in collagen 4

Purifying, detecting, and characterizing proteins

A protein must be purified to determine its structure and mechanism of action

Molecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties

SDS-polyacrylaminde gel electrophoresis

(SDS-PAGE)

Two-dimensional electrophoresis

immunoblotting

Pulse-chase exp

To investigate the fate of a specific newly synthesized protein

Cell + isotope for 0.5h

↓ wash

Different time point

↓

Immunoprecipitation

↓

Specific protein

↓

SDS-PAGE

↓

degrade

Low density lipoprotein receptor

<0.5h, protein convert to mature

PTM Glyco..

脈搏 補捉

Mass spectrometry can determine the mass and sequence of proteins

MALDI-TOF

Mass spectrometry can determine the mass and sequence of proteins

X-ray crystallography is used to determine protein structure

Figure 3-49

Other techniques such as cryoelectron microscopy and NMR

spectroscopy may be used to solve the structures of certain types of proteins

Advance technique in mass spectrometry are critical to proteomic analysis

Density-gradinet centrifugation and LC-MS/MS can be used to identify many of the protein in organelle

ATPase domain (homology with actin,

which also binds ATP)

Polypeptide binding domain with bound peptide ‘substrate’

Structure of entire molecule (~70 kDa) has not been solved

flexible linkage between ATPase and peptide-binding domains, and different

conformations of molecule possible

polypeptide-binding domain consists of beta-sheet scaffold;

loops possess hydrophobic residues that contact peptide

domain also has an alpha- helical ‘lid’ that is regulated by the ATPase activity

Structure of Hsp70 chaperone

Structure of Hsp70 chaperone

Major chaperones

and their interactions with substrates

?

Cellular processes involving Cellular processes involving non non - - native proteins: native proteins: refolding refolding

cellular stress

Native protein

non-native (unfolded)

protein heat/cold

proteotoxic chemicals intracellular

changes

aggregated protein

various cellular proteins

Summary of chaperon

Different protein → different function

Peptide segments of some protein are removed after synthesis Protein targeting/localization signals

• Signal peptide

• Mitochondrial targeting peptide

• Chloroplast targeting peptide

• Peroxisomal targeting signal (PTS2)

• Signal anchor

• Nuclear localization signal

• ER/Golgi retention signal

• Peroxisomal targeting signal (PTS1)

• Transmembrane helices

Cleaved

Uncleaved

Signal peptide or propeptide

N

Signal peptide

Propeptide

Mature protein

Characteristics of signal peptides

Length n-region h-region c-region -3, -1

Euk 22 only slightly Arg-rich

short, very hydrophobic

short, no pattern

small and neutral residues Gram- 25 Lys+Arg-rich slightly longer,

less hydrophobic

short, Ser+Ala-

rich

almost exclusively

Ala

Gram+ 32 Lys+Arg-rich very long, less hydrophobic

longer, Thr+Pro-

rich

almost exclusively

Ala

Ubiquitin–Mediated Proteolysis in Cellular Processes

Regulation of:

• Cell cycle

• Differentiation & development

• Extracellular effectors

• Cell surface receptors & ion channels

• DNA repair

• Immune and inflammatory responses

• Biogenesis organelles

Proteins Targeted by Ubiquitin

• Cell cycle regulators

• Tumor suppressors & growth modulators

• Transcriptional activators & inhibitors

• Cell surface receptors

• Mutant and damaged proteins

UbUb

E1 E1

UbUb

E2 E2

UbUb

E3 E3

UbUb

26s proteosome degradation 26s proteosome degradation

Target Target

The Ubiquitin Modification Pathway

UbUb UbUb

UbUb

E1 : Ub-activation enzymes. Modify Ub so that it is in a reactive state.

E2 : Ub-conjugating enzymes. Actually catalyze the attachment of Ub to the substrate protein.

E3 : Ub-ligases. Usually function in concert w/ E2. Play a role in recognizing the substrate protein.

Ubiquitin Conjugation:

A 3 Step Mechanism

Ubiquitin (Ub) activating enzyme E

High energy thiol ester is

formed between C-terminal Gly of ubiqutin and a Cys in the E

active site (ATP/AMP)

Ubiquitin conjugating enzymes E

Ub is transferred to a Cys of E

forming a new thiol ester

Ubiquitin ligase E

Ub forms isopeptide bond

between C-terminal Gly of Ub

and ε-amino group of Lys on a

target protein

The life of protein

Life cycle of a protein

Beta-amyloid Plaques

Amyloid precursor protein (APP) is the precursor to amyloid plaque.

1. APP sticks through the neuron membrane.

2. Enzymes cut the APP into fragments of protein, including beta-amyloid.

3. Beta-amyloid fragments come together in clumps to form plaques.

Enzyme are highly efficient and specific catalysts

A reaction will take place spontaneously only if the total G of the products is less than that of reactants.

All chemical reactions→high energy transition state→ rate of reaction is inversely to G → So need enzyme for catalysts

Enzyme: formed form protein

Highly efficient and specific catalysts

An enzyme active site binds substrates and carries out catalysis Active site: specific and chemical reaction site

Protein kinase A and conformational change induced by substrate binding rich

No ATP

Mechanism of phosphorylation by protein kinase A

Structure and function of pyruvate dehydrogenase, a large

multimeric enzyme complex that converts pyruvate into acetyl CoA

Molecular motors and mechanical work of cells

Motor proteins (mechanochemical enzymes): generate the forces necessary for many cellular movements, cells depend on specialized enzymes commonly called motor proteins.

Motion types: 1. linear; 2. rotor

Needs energy into motion

Three general properties of the activities of motor proteins:

1. Transduce a source of energy (ATP or ion gradient) for two types movement 2. Bind and translocate along a

cytoskeletal filament, nucleic acids strand or protein complex

3. Move direction

Motor protein-dependent movement of cargo

(protein, DNA, RNA..) Head: myosin, dynein, kinesin motor protein

All myosins have head, neck, and tail domains with distinct functions

Conformational changes in the myosin head couple ATP hydrolysis to movement

May be other cargo

1. Binding ATP → disrupting actin-binding site

2. Hydrolysis ATP → head, conformational change → move to new position → rebind

3. Pi release → head

conformation second change

→ move the actin

4. Release ATP → new cycle

flash

Functional design of proteins

Protein function generally involves conformational changes Proteins are designed to bind a range of molecules (ligands)

– Binding is characterized by two properties: affinity and specificity

Antibodies exhibit precise ligand-binding specificity Enzymes are highly efficient and specific catalysts

– An enzyme’s active site binds substrates and carries out catalysis

Mechanisms that regulate protein function

Allosteric transitions

– Release of catalytic subunits, active ⌦ inactive states, cooperative binding of ligands

Phosphorylation ⌦ dephosphorylation Proteolytic activation

Compartmentalization

Allostey: any change in a protein’s 3 or 4 structure or both induced by the binding of a ligand (activator, inhibitor substrate)

結合一個後→讓另一個更容易結合 (postive regulation) 釋放一個後→讓另一個結合力下降→更容易釋放

High affinity

Flexible

異位性調節

Allosteric protein – a protein in which the binding of a ligand to one site affects the binding properties of

another site on the same protein (also called induced fit model). The conformational changes induced by the modulator(s

modulator(s)) interconvert more-active and less-active forms of the protein.

allos --- other

stereos --- solid or shape

Homotropic interaction --- liagnd = modulator Heterotropic interaction --- ligand = modulator

O² --- as both a normal ligand and an activating homotropic modulator for Hb

The modulators for allosteric proteins may be either inhibitors or activators

Allosteric control: either an activator or inhibitor acts on a portion of the enzyme other than the active site to regulate enzyme function.

Allosteric transition between active

and inactive states

Ligand-indced activation of protein kinase Ligand binding can induce allosteric release of

catalytic subunits or transition to a state with different activity

Allosteric release of catalytic subunits

Switch mediated by Ca ²⁺/calmodulin In normal condition:

cytosolic calcium is low 10^-7 M by ER or pump.

ER release calcium to 10-100 fold → sense calmodulin →

conformal change → regulated other

protein or molecule

Ca ²⁺

Cycling of GTPase switch proteins between the active and inactive forms

Regulation of protein activity by

kinase/phosphatase switch

Purifying, detecting, and characterizing proteins

A protein must be purified to determine its structure and mechanism of action

Molecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties