Download MBP 1022, LECTURE 1 – Oct 27, 2000

Highlights of Chapters 1&2
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Key discoveries and Theories
Three Kingdoms
Cell and Genomes
Cell Chemistry
Fundamental questions
• What is the origin of life
• How does life propagate
• How can a single cell form a complex
organism
1859 Charles Darwin, Alfred Wallace
Evolution – origin of species - natural selection
fittest selected by forces of their environment
biological adaptation
Genes of different species are closely related
For instance some human genes will function in yeast and fly
Historical perspective of cell biology
1950-1960 – Golden age for cell/molecular biology
Fundamental breakthroughs – basis for todays
molecular understanding of biological systems
- Structure of DNA (stores genetic information, heredity)
- Central dogma (DNA
RNA
Protein)
- Genetic code (universal)
- Gene regulation (when, what and how much)
1980-Present – Information age of molecular biology
MOLECULES OF LIFE
Water – most abundant – 75-80% by wt
inorganic ions, small organic molecules
such as sugars, vitamins and fatty acids can be
made or imported
Macromolecules – protein, DNA, RNA, polysaccharides
must synthesize these
Proteins and DNA are polymers of monomeric units
amino acids for proteins (20)
nucleic acids for DNA (4)
proteins are the workhorses (proteins are versatile)
(enzymatic activity, structural proteins, transport)
DNA is the master molecule
Genetic analysis
(Inheritance of characteristics)
• 1865 Gregor Mendel – Pea plant
Important characteristics of his expts
– Pollination control easy
– Pure strains
– Defined characteristics
– Large sample size
YY
X
Yy
1865 Breeding Experiments
with Yellow & Green Pea seeds
yy
F1
Yy
X
Yy
YY
Yy
• Dominant/recessive
• 2 hereditary units (genes)
• Independent assortment (linked traits)
• One gene copy Allele
Yy
yy
F2
1953 – Modern Era of Molecular Biology
- Watson/Crick, Structure of DNA
double helix
- Chargoffs rules, G=C; T=A,
rules underlying the base pairing
- Wilkin/Franklin – X-ray diffraction pattern
helical nature, diameter, distance bet adjacent bp
RNA, genetic code
1959 – Crystal structure of protein
Structure
function relationships
Cell structure – Electron microscope, cell culture
1961 - Jacob and Monod – Regulation of gene
1950 - 60 - establishment of cell culture
Protein sequencing
1970 identification of specific restriction enzymes
dawn of cut and paste molecular genetics
advent of rapid DNA sequencing
oligonucleotide (DNA) synthesis
1980 Polymerase chain reaction
1990 Genome sequencing
Functional genomics
Systems analysis
Proteomics
Three animal Kingdoms
Eukarya
Bacteria
Archaea
Common single cell progenitor
Based on DNA sequence similarity Archaea
are more related to humans than bacteria.
Prokaryotes
•Prokaryotes
DNA is not sequestered
Simple internal organization
Eukaryotes
•Eukaryotes
Have a nucleus – compartment for DNA
organelles
Cells are small
Proteins are even smaller
Cell volume= 3.4 X 10-9 ml
Weighs 3.5 X 10-9 grams
20% protein 7 X 10-10 grams
Average protein size 52,700 grams/mol
7.9 X 109 proteins/cell
10,000 different proteins in cell
Suggests that there are over a million copies of each protein.
However, levels of certain proteins are tightly controlled.
Insulin receptor 20,000 copies per cell actin 5 X 108 copies
Many proteins within the cell are enzymes
Problem: How do cells keep inside
water in and keep outside water out?
All cells are surrounded in a lipid membrane
What other function can membranes serve?
Compartmentalize intracellular chemical reactions
Organelles
Mitrocondria-power plants
Endoplasmic reticulum-place to
make membrane proteins
and secreted proteins and lipids
Golgi vessicles-further refine
membrane proteins
and direct their transport to
specific surfaces of the cell
Peroxisomes-remove fatty acids,
hydrogen peroxide and
amino acids
Lysosomes-degrade old
proteins and foreign materials
The Superstructure of the Cell
Blue: DNA
Red: actin
cytoskeleton
Green: tubulin
cytoskeleton
DNA
4 nucleotides based-paired G=C, A=T.
Watson and Crick solved structure.
DNA strand coiled around a common axis forming a double helix
Flow of genetic information
Advent of genetic organization
Chromosomes
resides in the nucleus
means by which genetic information is transferred
number and size are constant in an organism
each chromosome – single DNA molecule (plus proteins)
can be considered a string of genes
total DNA – genome
visible during cell division
Somatic cells – diploid (2n), homologous pairs (mitosis)
Germ cells – haploid (n) only one of each pair (meiosis)
fruit fly (Drosophila) – 4; corn – 10; peas – 7; humans – 23
Chromosome
One human cell has 2 m of DNA found in 46 chromosomes
packed into a 0.006 mm3 nucleus
Chemical nature of the gene
Arranged as regular linear arrays
Gene order could change
Gene activity
Biochemical activity
One gene - One protein
S
DNA
R
S
contains all information
subject to variation/random change
faithful reproduction (like begets like)
underlies development of every new organism
LIFE CYCLE OF CELLS
• Steady state system in adult organism
balanced system (no net growth)
DNA
Proteins (maintenance)
DNA replication
Cell division
Cell differentiation
Cell apoptosis
Normal cell turnover
RBC
nerve cells
reproductive tissues
The Cell cycle
Cell Cycle follows a regular timing mechanism
Eukaryotes; Prokaryotes have no G0
Cell division 10-20 hrs vs 20-30 min
M – mitosis
G1 – first gap
S - synthesis
G2 – second gap
G0 – growth arrest
checkpoints
Mitosis
Mitosis – Partitions genome equally at cell division
Prophase, metaphase, anaphase, telophase
Cytokinesis, mitotic apparatus
Mitosis
(go to movies)
Meiosis
Cell Death/Apoptosis/Programmed cell death/Anoikis
• Balances cell growth multiplication
• eliminates unnecessary cells
(development, restructuring, damaged cells)
• internal program (clock)
• follows systematic events (DNA frag, membran
blebbing, consumed by macrophages)
• Now an important area of cancer research
Cells are organized into Tissues
• Extracellular matrix (ECM)
network of proteins and polysaccharides
• Cell-adhesion molecules
cell-cell contact
cell-ECM contact
• basal lamina
• endothelium
Body Patterning dictated by patterning genes
• program of genes specify the body plan
• local interactions induce specific program
• Conserved throughout evolution
• axial symmetry
• integration / coordination of multiple events during
embryogenesis
1
genetic program
2
cell contact
gene expression
adhesion
3
soluble factors
signaling
Cell Differentiation – 200 different cell types in the body
Change to carry out a special function
Marked by a change in morphology
“form follows function”
(examples are nerve cell vs muscle cell)
creates diversity of cell types required
Examples: fertilized eggs
Organism
stem cell
heart & vessels
Power of DNA to orchestrate cellular change
Heart Development Requires Proper Vessel Growth and
Differntiation of many different cell types
CHAPTER 2 – Cell Chemistry and Biosynthesis
Chemical concepts underlying cellular processes
Basic principles of chemistry and physics direct
biological processes.
No supernatural force is required for biological processes
BONDS and STABILIZING FORCES
CHEMICAL EQUILIBRIUM
ENERGY
CENTRAL ROLE OF ATP
ENZYMES
WATER – constitutes 70-80% ; small molecules ~ 7%
Rest - MACROMOLECULES
BUILDING BLOCKS
Amino acids
Proteins
Nucleotides
DNA and RNA
Sugars
Complex Carbohydrates
CHEMICAL BONDS
Covalent (50-200)
Noncovalent (1-5 kcal/mole)
- Strong
- Weak
- sharing electrons
- 3D structure
within atoms of an
- inter and intra molecular
individual molecule
- Strength – cooperation
Orbitals
- multiple, weak bonds
- transient, dynamic
Nucleus
protons
Electrons
Covalent Bonds
A. Atoms in biological systems
• Hold the atoms within a molecule
• Formed by sharing electrons in the outer atomic orbitals
• Forms the basis of chemical reactivity and basic shape
H
C
1
4
N
P
O
S
3
5
2
2,6
• Each atom can make a defined # of covalent bonds
• Depends on the number of electron in the outermost
orbital and their size
• typically stable (making/breaking bonds requires energy)
• energy required to break a single bond (50-100 kcal/mol)
double bond (120-170 kcal/mol); triple (195 “)
Examples:
- phosphorous – biologically very important
- esters of sulfuric acid – proteoglycans in ECM
B. Bonds are oriented at precise angles (shape)
H
O
104.5 (water, each single bond)
H
• dependent upon mutual repulsion of outer e orbitals
• non-bonding electrons also contribute to properties/shape
• double bond are more rigid (cannot rotate freely)
D. Asymmetric carbon (common in biological molecules)
a carbon atom bonded to four dissimilar atoms
COOH
COOH
H - C - NH2
NH2 -C - H
CH3
D-alanine
CH3
mirror image
L-alanine
• Optical isomers (stereoisomers) designated D or L
• Central C is called chiral carbon (alpha C)
• All naturally occurring aa in proteins are L.
• only D form of sugars (carbohydrates are found)
• different biological activity, but identical chemical property
NON COVALENT BONDS or INTERACTIONS
• Hydrogen bond
• Ionic Interactions
• van der Waals Interactions
• Hydrophobic bond
Important for stabilizing 3D structures
Inter- and Intra-molecular
Multiple bonds give strength
Transient/dynamic
A. Hydrogen Bond (~ 5 kcal/mol)
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•
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•
•
•
•
•
•
Underlies chemical and biological property of water
When H atom covalently bonded to another atom (donor,D)
forms a weak association (the hydrogen bond) with an
acceptor (A) atom
Both D, A – electronegative and polar
Most D, A are N (3.0) or O (3.4)
N-H
C-H
polar
nonpolar
O-H
Forms the basis of solubility (hydrophilic – water loving)
More H bonds, more soluble
Standard length (0.26-0.31 nm) and directionality (linear/strong)
Stabilizing force is multiplicity
H bonding usually involves exclusion of a H2O molecule
B. IONIC INTERACTIONS
• When bonded atoms have very different electronegativilty
• e- found among more electronegative atom (Na+Cl-)
• no fixed orientation/angel
+vely charged ion (Cation)
_vely
charged (Anion)
Na+, K+, Ca2+, Mg2+, Cl• typically exist complexed to H2O (using the water dipole)
• important biological roles (nerve impulses, muscle contraction)
• very soluble and energy is released as they bind water
• energy of hydration
C. Van der Waals Interactions (~ 1kcal/mol)
• non-specific attractive force is created as two
atoms approach each other closely
• transient / momentary fluctuations in the distribution of e
generating a transient electric dipole
• seen in all types of molecules (polar and non-polar**)
• H bonds, ionic interactions can override VDW
• Van der Waal radii – balance attraction
repulsion
• antigen:antibody / enzyme:substrate
facilitated by their complementary shape
D. HYDROPHOBIC BONDING (force that causes hydrophobic
molecules to aggregate rather than dissolve)
• non-polar molecules (for example hydrocarbons)
• no ions, no dipole moment, no hydration
• Force that causes non-polar molecules to aggregate
• Basic force for BIOMEMBRANE structure
A phospholipid bilayer typically separates two aqueous
compartments (plasma membrane and organelle memb)
• Phospholipids are amphipathic (tolerant of both) molecules
Fatty acyl chains – glycerol – phosphate – alcohol
Hydrophobic
Hydrophilic
Orient their hydrophilic ends to
The aqueous environment
Spontaneously organize into structures (micelle, liposomes, bilayer)
Impermeable to salt, sugar and small molecules
VdW interactions stabilize the close packing
This structure is very fluid
Proteins – span the phospholipid bilayer
CHEMICAL REACTIONS
•
•
•
covalent bonds are broken and re-formed
several hundred different rxns may occur simultaneously
in a given cell
what rxns can proceed (rate/extent) depend on multiple
factors
1. concentration of reactants (initial determinant)
2. catalyst
3. pH, pressure, temperature
Chemical Equilibrium: is reached when the rates of
forward and reverse reactions are equal.
A+B
Keq
X+Y
= [X] [Y]
[A] [B]
Equilibrium constant is the ratio of products to reactants
A catalyst can increase the rate of reaction.
pH: Concentration of positively charged (H+) ions
• dissociation products of H2O (H+, OH-) are constantly
liberated
• when H+ is produced, it combines with a H2O molecule
(hydronium ion - H3O+)
• dissociation of water is a reversible rxn
H2O
@ 25o C
In pure water
H+ + OH[H] [OH] = 10-14 M2
[H] = [OH] = 10-7M
•pH = -log [H] = log 1
[H+]
•In pure water @ 25o C, [H+] = 10-7 M
•pH = -log 10-7 = 7 (Neutral)
higher value than 7 is basic;
lower than 7 is acidic
•pH – is an important property of a biological fluid
•Different cellular organelles have selective pH
•Maintenance of precise pH is imperative for cellular function
•Change in pH – a way of controlling cell activity
ACIDS and BASES
- Acid, any molecule that releases H+
- Base, any molecule that combines with H+
- organic molecules are acidic (COOH) produce COOO
X-COOH
X-C
X-C
H
X-NH2 + H+
O
+ H+
O-
X-NH+3
- Whenever add acid, increase in H+
add base, increase in OH- or decrease in H+
- All solutions contain some H and OH
-Biological molecules can have both acidic and basic groups
-pH determines the degree to which H/OH groups are released
COO-
COOH
H - C - NH2
@ pH 7.0
H - C – NH3+
R
R
Zwitter Ions (neutral)
Doubly ionized form
Amino acid
pH
COOH
H - C – NH3+
R
pH
COOH - C – NH2
R
Molecules have multiple acidic/basic groups
[H+] + [A-]
HA
Ka
=
[H+] [A-]
[HA]
log Ka =
log [H+] + log [A-]
[HA]
pH
pKa
=
+ log [A-]
[HA]
pKa is the pH at which 50% of molecules are
dissociated, the other 50% being neutral
(Henderson Hasselbalch Equation)
• pH must be maintained near 7.2 in the cell cytoplasm
• buffers are weak acids or bases
(soak up [H+] and [OH-] ions
• ability of a buffer to minimize the change in pH (buffering capacity)
• pKa shows the buffering capacity
Example is phosphoric acid (3 groups capable of dissociating)
O
=
H3PO4
HO – P – OH
OH
H2PO4- + H+
pKa = 2.1
H PO42- + H+ pKa = 7.2
PO43- + H+
pKa = 12.7
Physiologically important buffer (cytosol pH 7.2, blood 7.4)
ENERGY – defined as the ability to do work
•
Kinetic (the energy of movement)
- Heat/thermal; Radiant- photons**; Electric - electrons
• Potential (stored energy)
- Chemical bonds; Concentration gradient; Electric
potential
- Important in biological systems
- Glucose is the central molecule
•
The law of thermodynamics:
- Energy is neither created nor destroyed
- converted from one form to another
- Unit: Calorie (cal) = 4.18 Joules
1000 cals = 1kcal
ATP – Adenosine triphosphate (Ap~p~p)
(the cellular currency for energy)
O
O
O
=
=
=
O- – P- O – P- O – P – O – H2C
O-
O-
Base
O-
Sugar
Phosphoanhydride bonds
(High energy bonds) = -7.3 kcal/mole, moderate
Package (easy to make, can drive many rxns)
• Captures and transfers energy
• used to transfer P to one of the reactants
(high energy intermediate)
• difference in energy released from ATP vs AMP
WHAT CAN THE ATP BE USED FOR:
• macromolecular synthesis
• cell movement (muscle contraction)
• transport molecule in/out cell
• generate concentration gradients
• generate electric potential (nerve impulse)
ENZYMES:
• straining of covalent bonds
• excitation of e• overcome mutual repulsion of e- cloud
• In biological systems kinetic energy of colliiding
molecules is insufficient
• act primarily by reducing the activation energy
• facilitate movement of H atoms / e- / protons
• strain bonds and stabilize transition state
• formation of covalent bonds
• Proteins, highly specific substrates
• catalysts do not change themselves