Transcript Structures define the functions of proteins

Announcement
Advanced Molecular Biology course 2015 is Essential
(1) Syllabus
Two parts (Dr Cho, Dr Kim)
First part covers three themes
- Protein Structure and Function (Ch3)
- RTK strcuture and anti-cancer monoclonal antibody
drugs
- Biophysical techniques for experiments
(2) How to get lecture slides
structure.yonsei.ac.kr/
File name: AMB_Ch3
Announcement (continued)
(3) Exam and Grading
2 times (Dr Cho (45%), Dr Kim (45%), attendance(10%) )
 Problem types: Short or long answer with figures100%
 Place: Lecture Room S118A
 Posting of score in Exam: on the board at room SB134,
(4) Interviewing with me
 You may see me during this course if you want
 My office hours: AM 10:00-11:00 on Thursday
 How: First, Contact me by E-mail or telephone
 E-mail address: [email protected], 2123-5651
Harvey Lodish • Arnold Berk • Paul Matsudaira •
Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell
Molecular Cell Biology
Fifth Edition
Chapter 3:
Protein Structure and Function
Copyright © 2004 by W. H. Freeman & Company
Protein Structure and Function
•Function is derived from three-dimensional structure
;
- Structures define the functions of proteins
•Only when a protein is in its correct threedimensional structure, it is able to function
F1-ATPase
Principles of Biology
•Living organisms are subject to basic laws of
chemistry and physics
• Major difference between general chemistry and
biology; they include more than 70% waters. so
biological molecules are always surrounded by waters
•
Four of water’s properties for life
– Cohesive behavior
– High heat capacity
– Expansion upon freezing
– Versatility as a solvent
• H-bonds
• Peptide bond is plane!
• the size of protein; dalton
• average MW of amino acids in protein : 113
Secondary structure : alpha helix, beta sheet
60% polypeptide chain, H-bond
Alpha helix :
(n, n+4) carbonyl oxygen, amide hydrogen atom
Direction : where is the N-terminal ?
Top view
Side view
Beta sheet :
Parallel, antiparallel
pleated sheet
Turns : 3-4 residues, glycine, proline common
Motifs : paticular Combinations of Secondary Structures
Structural and functional domains are modules of tertiary structure
Heamagglutinin –
Surface protein in influenza virus
Structural domain : proline-rich domain, acidic domain, SH3, zinc-finger motif
Functional domain : kinase domain, DNA-binding domain
Epidermal growth factor
Proteins Associate into multimeric structures and Macromelecular assemblies
RNA polymerase II : 12 subunits
(Dr. Roger kornberg)
Mediator : 20 subunits
Members of protein families have a common evolutionary ancestor
4 subnits
* Oxygen-binding globin
Plant
Blood
Homologous proteins belongs to a family
Non-homologous protein : similar structure, similar function
Muscle
Folding, Modification, and Degradation of proteins
• The information for protein folding is encoded in the amino acid sequence
• Folding of proteins in vivo is promoted by chaperone
95% proteins within cells are in native conformation despite of high
concentration (200-300mg/ml), which favor the precification of proteins in
vitro.
This can be explained by chaperones. (above 85% protein folding)
- Molecular chaperone : bind to exposed hydrophobic regions and stabilize them,
thereby preventing these proteins from aggregating and being degraded.
(Hsp70+Hsp40, GrpE+DnaK)
- Chaperonins directly facilitate the folding of proteins
(TriC, GroEL)
ATP X, ADP binding ;
bind to misfolded protein
ATP binding, GroES ;
Releases the folded protein
Cavity twofold increase
TriC
Chemical Modification of amino
acid residues
Nearly every protein in a cell is chemically modified after its synthesis.
This may alter the activity, life span, or cellular location of proteins.
80% proteins are acetylated ; lifetime control,
Nonacetylated protein – short lifetime
collagen
Membrane receptors
prothrombin
Phosporylation : Serine, threonine, tyrosine, histidine
Glycosylation : asparagine, serine, threonine
Lipid attachment :
Repression by nucleosomes
Coiling of DNA around a histone octamer in the nucleosome is
now recognized as a cornerstone of transcriptional control.
Nucleosomes repress transcription in at least three different ways.
First, they occlude sites of protein binding to DNA, thereby interfering
with the interaction of activator and repressor proteins,
polymerases and transcription factors, DNA-modifying enzymes.
Second, chains of nucleosomes can become further
coiled or folded, and this higher-order coiling represses transcription
of entire chromosomal domains.
Finally, interactions of
nucleosomes with additional chromosomal proteins in heterochromatin
repress gene expression in a hereditary manner6.
Each histone is organized
in two domains, a characteristic ‘histone fold’ and an unstructured
N-terminal ‘tail’. The histone-fold domains constrain the
DNA in a central core particle and, thereby, restrict access of
DNA-binding proteins.
This histone tail is a flexible amino terminus of 11-37 residues.
Several positively charged lysine side chains in the histone tail may
Interact with linker DNA, and the tails of one nucleosome likely interact with
Neighboring nucleosomes  higher-order coiling.
The histone tail lysine, especially those in H3 and H4, undergo reversible
acetylation and deacetylation by enzymes such as CBP (P300) and HDACs
In the acetylated form, the positve charge of the lysine e-amino group is
neuralized. This eliminate its interaction with a DNA phosphate group.
So the greater the acetylation of histone N-terminus, the less likely chromatin
is to form condensed 30-nm fibers and possibly higher-order folded
structures.
Sites of Histone Tail Modifications
Epigenetics edited by Allis et al. (2007)
Distinction between Euchromatic and Heterochromatic Domains
Euchromatic
Hallmarks
H3K4-Me
Heterochromatic
Hallmarks
H3K9-Me,
H3K27Me
Epigenetics edited by Allis et al. (2007)
Proteolytic cleavage : blood coagulation, digestion and apoptosis
EGF, insulin
Protein self-splicing : internal segment is removed. Hedgehog.
Serine is part of a catalytic triad that also includes histidine and aspartate
-The three-dimensional structure
of chymotrypsin was solved by
David Blow in 1967.
-It is synthesized as a single
polypeptide, termed
Chymotrypsinogen, which is
activated by the proteolytic
cleavage to yield the three chains.
[1GCT.pdb]
cleft
-The active site of chymotrypsin, marked by serine 195, lies in a
cleft on the surface of the enzyme.
-This side chain of serine 195 is hydrogen bonded to the imidazole
ring of histidine 57.
Tetrahedral
intermediate
(acyl-enzyme)
Nucleophilic attack
Amine is free
Burst phase
Substrate binding
Steady-state phase
Carboxylic acid
product
Water mediated
deacylation
OH- Attacks the
carbonyl carbon
Degradation of protein
Lysosomal pathway : extracellular proteins, hydrolytic enzyme and acidic sol’n
Ubiquitination : lysine residue attached 76-residue peptide.
E1,E2 : thioester bond
E3 : specific substrate binding protein
isopeptide bond
Cytosolic protein ; cyclin
Misfolded in ER
Immune system
N-terminal rule : stabilizing (Met, pro),
destabilizing (Arg, leu)
E3 enzyme read N-terminal residue
; cell cyclin (internal sequence)
misfolded protein (hydrophobic sequence)
26S proteasome
• 20S proteasome (CP) : 700kD, catalytic (7-9 a.a)
• 19S proteasome (RP) : 700kD, 6 ATPase+isopeptidase
Digestive Proteases Degrade Dietary Proteins
Zymogen,
stomoch (pepsin, F,L), pancrease (trypsin, chymotrypsine; basic, aromatic)
Misfolding not only leads to a loss of the normal function of the protein but also
Marks it for proteolytic degradation.
The proteolytic fragments  filamentous plaques
degenerative disease ;
Alzheimer’s disease, Parkinson’s disease, mad cow disease
Amyloid precursor  beta amyloid
(a helix  b sheet)
MW ?
Kd ?
High shap complementarity :