Molecular Cell Biology
Fifth Edition
Chapter 3:
Protein Structure and Function
Copyright © 2004 by W. H. Freeman & Company
Harvey Lodish • Arnold Berk • Paul Matsudaira • Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell
F1-ATPase:
carries out the
synthesis of ATP
in chloroplasts,
and mitochondria.
Functional classes:
Structural proteins Transport proteins Regulatory proteins Motor proteins
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
H
3N C COO H
R
pK1 pK2
+ -
1o 2 o 3o 4o 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
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
Peptide bond- linkage between amino acids is a secondaryamide bond Formed by condensation of the α-carboxyl of one amino acid with the α-amino
of another amino acid (loss of H2O 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-C N 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
Secondary structure: the beta sheet
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
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
04_05_Hydrophobic.jpg
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
一致
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
0) 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
Different protein → different function
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
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
Folding, modification, and degradation of proteins
The information for protein folding is encoded in the sequence Conformational folding can denature to polypeptides
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
• 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
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)
Chperonins (GroEL/ES) in protein folding
Protein folding
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
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 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
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
Ub Ub E1 E1
Ub Ub E2 E2
Ub Ub E3 E3
Ub Ub Target Target
The Ubiquitin Modification Pathway
Ub Ub Ub Ub
Ub Ub
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
Ubiquitin Conjugation:
A 3 Step Mechanism
Ubiquitin (Ub) activating enzyme E
1High energy thiol ester is
formed between C-terminal Gly of ubiqutin and a Cys in the E
1active site (ATP/AMP)
Ubiquitin conjugating enzymes E
2Ub is transferred to a Cys of E
2forming a new thiol ester
Ubiquitin ligase E
3Ub forms isopeptide bond between C-terminal Gly of Ub and ε-amino group of Lys on a target protein
THE PROTEASOME
The life of protein
Life cycle of a protein
Digestive proteases degrade dietary protein
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.
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.
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
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
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
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
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)
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
O
2--- 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
-7M 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
Schematic of membrane proteins in a lipid
bilayer
Membrane proteins
Each cell membrane has a set of specific membrane proteins that allows the membrane to carry out its distinctive activities
Membrane proteins are either integral or peripheral
Integral transmembrane proteins contain one or more transmembraneα helices
Other integral proteins are anchored to the membrane by covalently attached hydrocarbon chains
Peripheral proteins are associated with membranes through interactions with integral proteins
Integral membrane proteins can be solubilized by non- ionic detergents
Figure 3-39
Centrifugation can separate molecules that differ in mass or density
Electrophoresis separates molecules according to their charge:mass ratio
SDS-polyacrylamide gel electrophoresis
SDS-gel electrophoresis
Two-dimensional electrophoresis separates molecules
according to their charge and their mass
Separation of proteins by size: gel filtration
chromatography
Separation of proteins by charge: ion exchange chromatography
Separation of proteins by specific binding to another molecule:
affinity chromatography
Highly specific enzymes and antibody assays can detect individual proteins
Protein primary structure can be determined by chemical methods and from gene sequences
Edman degradation
Time-of-flight mass spectrometry measures the mass of proteins and
peptides
Figure 3-47
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
