Transcript Center for Structural Biology

Distinguishing Features
Separate the protein from all other contents
Sequence and fold give overall properties
Molecular weight
Solubility
Exposed hydrophobic surface
Ability to bind other molecules, metals
pI- the overall charge of the protein
Sequence!!!
Protein Analysis
Purification
A. Simple solubility characteristics- precipitation
B. Chromatography- ion exchange, size, hydrophobic,
specific affinity
C. Gel electrophoresis- native, denaturing (SDS)
Characterization
A. Sequencing- degradation, Mass Spectrometry
B. Spectroscopy- UV, CD, fluorescence, EPR, NMR
C. Antibody binding- specificity
D. Structure- X-ray crystallography, NMR
Proteins Function By Binding
• Transport- O2/CO2, cholesterol, metals, sugars
• Storage- metals, amino acids,
• Immune response- foreign matter (antigens)
• Receptors- regulatory proteins, transmitters
• Structure- other structural proteins
• Enzymes- substrates, inhibitors, co-factors
• Toxins- receptors
• Cell functions- proteins, metals, ions
Surface properties: steric access, shape, hydrophobic
accessible surface, electrostatic surface
Regulation of Protein Function
1. Allosteric Control
2. Stimulation/inhibition by control factors
3. Reversible covalent modification
4. Proteolytic activation/inactivation
Static Structure/Dynamic Biology
Structures are static snapshots of highly dynamic
molecular systems
Biological process occur at femtosec - min. timescale
NMR of Proteins
Challenges
Proteins have hundreds/thousands of signals
Resonance assignment first…..who do all these
signals belong to?
Need to use computer programs to convert from
NMR data to structures
Applications
Folded protein?
Measure binding constants
Assess structural homology/effect of mutations
Three-dimensional structure determination
Measure flexibility/dynamics
Enzymes: Protein Catalysts
 Increase rates of reaction, but not consumed
 Enable reactions to occur under mild conditions
 High reaction specificity/no side products
 Catalytic mechanisms: Bond Strain, Proximity/Orientation,
Acid/Base Catalysis, Covalent Catalysis, Metal Ions,
Electrostatic, Preferential Binding of the Transition State
 Activity of enzymes can be regulated
 Availability of substrate or enzyme
 Reversible covalent modification
 Allosteric control (other proteins or co-factors)
 Activity of enzymes can be inhibited
 Competitive inhibition
 Uncompetitive inhibition
 Mixed or non-competitive inhibition
 Inactivator
Energies and Rates of Reactions
 The transition state is the highest point on the
reaction coordinate diagram.
 The height of the energy barrier is the Free Energy of
Activation: DG‡.
 Backwards barrier usually higher than forward. The
difference in energy between substrate and product
is the Free Energy of Reaction.
 If the energy barrier is higher for one step than the
other, than the rate of this step will be slower. The
step with the highest transition state free energy (the
highest point on the reaction coordinate) is the Rate
Determining Step of the reaction.
KEY POINT: Ref. Is DG‡
The Catalytic Effect of Enzymes
 Enzymes lower transition state, reducing DG‡
 The catalytic efficiency (DDG‡cat) is DDG‡ catalyzed vs.
uncatalyzed
 Catalytic efficiency reflected in kinetic parameters: rate
enhancement for the reaction.
 Lowering of the free energy barrier and increase in rate
is equal for the forward and the reverse reactionstransition state energy is lowered!!!
 Time required to come to equilibrium is less.
 Increase in velocity with which products are produced
from reactants and vice versa.
 No change in the ratio product:substrate: DGreaction
First Order Rate Equations (cont.)
Equation for a first order reaction: ln[S] = ln[S]o –kt
A plot of ln[S] vs. t is a straight line:
 The intercept is the starting concentration [S]o
 Slope is the negative of the rate constant (k)
Half-life of the reaction (t½) is the time required for half of
S to be used up. The slope of the line in the plot never
changes and the half-life is the same regardless of the
starting concentration.
t½ = ln2/k = 0.693/k
Enzyme Kinetics
E + S  ES  P + E
E- enzyme, S- substrate, ES- enzyme-substrate complex,
P-product, k1,k2 - forward rates, k-1 - reverse rate, k-2 negligible)
v = d[P]/dt = k2 [ES]
[ES] is difficult to measure because it is time dependent:
d[ES]/dt = k1 [E][S] - k-1[ES] - k2[ES]
Only the total concentration of enzyme [E]T = [E] + [ES]
can be measured.
Steady State Assumption
 At steady state: [S] >> [E], [ES] remains constant
because all enzyme active sites filled with S,
d[ES]/dt = 0
 Recall from the last page:
d[ES]/dt = k1 [E][S] - k-1[ES] - k2[ES]
v = d[P]/dt = k2 [ES]
[E]T = [E] + [ES]
 So, at steady state:
v = d[P]/dt = k2 [ES] = k1[E] [S] - k-1[ES]
k1 term
k1 [E] [S] = k-1 [ES] + k2 [ES] = (k-1 + k2) [ES]
substitute
k1 ([E]T - [ES]) [S] = (k-1 + k2) [ES]
Continuing to Reorganize
k1([E]T - [ES]) [S] = (k-1 + k2) [ES]
shift k1
([E]T - [ES]) [S] / [ES] = (k-1 + k2) / k1
Define the Michaelis Constant (KM) = (k-1 + k2)/k1
shift [ES]
([E]T - [ES]) [S] = KM [ES]
expand
[E]T [S] - [ES] [S] = KM [ES]
swap term
[E]T [S] = [ES] [S] + KM [ES] = ([S] + KM) [ES]
reorganize
[ES] = [E]T [S] / KM + [S]
Express With Measurable Quantities
[ES] = [E]T [S] / KM + [S]
Using this relationship, and working in the regime where
the back reaction for [ES] is negligible, the initial velocity
can be expressed in [ET] and [S], which are measurable
quantitites!
vo = k2 [ES]
 vo = k2 [E]T [S]/(KM + [S])
At saturation [E]T = [ES], so:
vo = Vmax = k2 [E]T
This gives Michaelis-Menten Equation of enzyme kinetics:
vo = Vmax [S] / (KM + [S])
How to Characterize M-M Kinetics
 M-M Equation:
[S] << KM,
[S] = KM,
[S] >> Km,
vo = Vmax [S] / (KM + [S])
vo = (Vmax / KM ) [S]
vo = Vmax /2
vo = Vmax
 Define kcat, turnover number:
kcat = Vmax / [E]T
When [S] << KM, very little ES is formed, so [E]T = [E]
vo ~ (kcat / KM) [E] [S]
Implications of M-M Equation (cont.)
The kcat/Km term is a measure of the enzyme’s catalytic
efficiency: how often a molecule of substrate that is
bound reacts to give product
The upper limit to kcat/Km is k1 because decomposition of
ES to E + P can occur no more frequently than ES is
formed.
The most efficient enzymes have kcat/Km values near the
diffusion-controlled limit: conditions where a reaction
occurs almost every time a substrate is bound.