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Introduction into Cell Biology 2 The building blocks of life - Proteins

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Title: Introduction into Cell Biology 2 The building blocks of life - Proteins


1
Introduction into Cell Biology 2 The building
blocks of life - Proteins
2
Intro into cell biology 2
Molecular Organisation of a cell
3
Fig. 1.7
4
Building Blocks of Life -gt Different Shapes
5
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6
Proteins Amino Acids are linked by peptide bonds
7
20 Natural Occurring Amino Acids are divided
into groups according to their side chains
8
The Aromatic Amino Acids
9
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10
Cys can cross-link between 2 polypeptide chains
-gt Disulfide bridge
11
Proteins are Polypeptides
Direction of a Protein
12
3D Structure of Proteins
13
Interactions between side chains and backbone -gt
Fold of a protein (3D structure)
14
Noncovalent interactions within and between
biological molecules
15
Secondary Structure
1. a Helix 2. ß-Strands -gt ß-Sheets 3. Loops
and Turns
16
a-Helix
17
Examples of a-Helical Proteins
Hair
18
Examples of a-Helical Proteins
Muscle
a-helical coiled coil proteins Form
superhelix Found in myosin, tropomyosin (muscle),
fibrin (blood clots), keratin (hair)
19
Examples of a-Helical Proteins
20
ß-Strands -gt ß-sheets
21
Examples of ß-sheet Proteins
Fatty acid binding protein -gt ß barrels structure
Antibodies
OmpX E. coli porin
22
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23
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24
Turns and Loops
Loops in Receptors
Turn
25
Tertiary Structure 3D structure of a polypeptide
chain
26
Quaternary Structure Polypeptide chains
assemble into multisubunit structures
Cell-surface receptor CD4
Tetramer of hemoglobin
27
Protein Folding
Folding is a highly cooperative process (all or
none)
Folding by stabilization of Intermediates
28
Protein Folding by Chaperons
29
Misfolded protein -gt Alzheimer
Protein fibrillation
30
Function of Proteins
31
Molecular Machines Transcription Initiation
Complex
32
Function of Proteins
Specific binding of ligands -gt Immunoglobins
33
Function of Proteins
Conformational change of lactoferrin upon binding
of Fe
Conformational change induced by Calcium
34
Function of Proteins
Activation by modification
GFP fluorescent Rearrangement and oxidation of
Ser-Tyr-Gly
35
Function of Proteins
Model of enzymatic reaction mechanism
36
Proteins Key properties
  1. Proteins are linear polymers built of Amino Acids
  2. Proteins contain many functional groups (i.e..
    side chain of AA)
  3. Proteins interact with proteins and with other
    biological molecules to form complexes
  4. Proteins can bind and/or modify other molecules
  5. Proteins can be rigid or can have regions with
    high flexibility

37
Enzyme Kinetics
  • Enzymes DO NOT shift the equilibria but enhance
    the rates of the reactions (lower the activiation
    energy!!!)

38
Transition state
  • Unstable state of maximum energy
  • Not an intermediate
  • Metastable state
  • Intermediates are species that appear in a
    reaction mechanism but not in the overall
    balanced equation.

DH
H
DH0
Reaction coordinate
39
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40
Reaction Kinetics
Thermodynamics does a reaction take
place? Kinetics how fast does a reaction
proceed?
Reaction rate is the change in the concentration
of a reactant or a product with time (M/s).
DA change in concentration of A over
time period Dt
DB change in concentration of B over
time period Dt
Because A decreases with time, DA is
negative.
13.1
41
13.1
42
Basic problem of enzyme kinetics
Suppose an enzyme were to react with a substrate,
giving a product.
43
Michaelis and Menten
In 1913, Michaelis and Menten proposed the
following mechanism for a saturating reaction rate
Complex.
product
44
Michaelis-Menten Kinetics
  • When S ltlt KM, the reaction increases linearly
    with S I.e. vo (Vmax / KM ) S
  • Very little ES is formed
  • When S KM, vo Vmax /2 (half maximal
    velocity) this is a definition of KM the
    concentration of substrate which gives ½ of Vmax.
    This means that low values of KM imply the enzyme
    achieves maximal catalytic efficiency at low S.
  • When S gtgt Km, vo Vmax

Where activity measurements should be performed
1. S very high

2. all enzyme bound in ES complex
45
Michaelis-Menten Kinetics
When the enzyme is saturated with substrate, the
reaction is progressing at its maximal velocity,
Vmax. Combing the steady-state assumption
(dES/dt0) with the conservation condition
(ETE ES) vo leads to the
Michaelis-Menten Equation of enzyme kinetics

where Km is

KM (k-1
k2)/k1
46
Michaelis-Menten Kinetics
What is Vmax and KM ?
  • KM gives an idea of the range of S at which a
    reaction will occur. The larger the KM, the
    WEAKER the binding affinity of enzyme for
    substrate.
  • Vmax gives an idea of how fast the reaction can
    occur under ideal circumstances.

47
Michaelis-Menten Kinetics
Determination of Enzyme kinetics -gt Measure
activity (velocity) at different substrate
concentrations Determine activity of an Enzyme
-gt Measure at substrate concentration of above
10KM -gt no substrate limitation ETES
48
Michaelis-Menten Kinetics
How to measure activity of an enzyme using
photometrical method ? Lambert Beer law
A c e
l Where A is the absorbance c is the
concentration (mol/L) e is the molar
absorption coefficient (L/mol cm) l
is the path length of sample (cm)
Rate activity ?c/?t -gt ?A/?t l e
?c/?t activity rate (unit)
-gt ?c/?t (?A/?t)/le Definition 1 Unit of an
enzyme will catalyse the reaction of 1 µmol of
substrate within 1 min at certian pH and
Temperature. Measure ?A/?t (change in
absorption/min) for a special enzyme and a high
substrate concentration -gt from that you can
calculate activity of that enzyme at special
Temp., solvent, pH, pressure.
49
Michaelis-Menten Kinetics
How do determine experimentally KM and Vmax ?
Lineweaver-Burk plot
Eadie-Hofstee plot
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