Lecture 4 - protein structure and function 1 Flashcards
what are structural proteins?
Determine cell shape and contribute to the extracellular environment. Include actin and tubulin - filaments that control movement within cells, movement of cells and define cell shape
Structural proteins can form molecular machines by self-assembly such as microfilaments, intermediate filaments and microtubules
what are scaffold proteins?
Bring proteins together into an ordered complex; allows increased efficiency than if the proteins were not assembled together
(eg when free diffusion is changed to be more efficient) can become a local adaptor to make a stimulus
what are enzymes?
they bind to a substrate to create a complex. this complex then produces a product, increasing enzyme activity then the product is released. speeding up a chemical reaction.
what are membrane transport proteins?
Membrane proteins responsible for moving material from one side of the membrane to the other. This can be solutes, ions, proteins or signals
what are regulatory proteins?
Signals, sensors and switches that control cell function by altering the function of other proteins.
These include receptors and signalling proteins, like the kinases lectured on in Fundamental Topics in Biology-2
what are motor proteins?
Motor proteins move other proteins, organelles, cells or even whole organisms (think actin/myosin in our muscles). Proteins do ‘work - they use chemical energy to beat entropy!
can proteins belong to more than one class of protein?
yes! proteins can belong to more than one of these classes.
A good example is the insulin receptor covered in Fundamental Topics in Biology-2; this is a receptor for a signal, it delivers that signal across the membrane and has a kinase activity. So its a membrane protein, a regulatory protein and an enzyme.
To accomplish their actions efficiently, many proteins assemble into large complexes, these are sometimes called ‘Molecular Machines’.
how do proteins perform such diverse functions?
-Most exploit a few basic activities:
Catalysis
-They bind (to other proteins, to DNA, to substrates…)
-Fold into specific shapes - conformations
-These are often linked events and regulate activity of the protein
what is binding
Binding involving proteins, either to each other or to ligands, can be quantified.
Binding can be controlled and binding can in turn control activity of a protein.
Binding is reversible!
This is important - consider an actin filament in your muscle, or an antibody binding a toxin in your blood or insulin binding to its receptor.
how do proteins bind?
Both inside and outside cells biomolecules collide. The higher the concentration of molecules, the greater the frequency of collision.
When two molecules collide, it is likely they will ‘bounce apart’ because the non-covalent interactions that might hold them together are weak and are transient under physiological conditions (pH, salt, temperature etc.)
However, molecules that exhibit molecular complementarity can form multiple non-covalent interactions at close range.
When such molecules collide, these multiple interactions cause them to stick together - bind
what does binding depend on?
Depends on the geometry of the two binding partners
NO other arrangement of atoms will produce as energetically favourable complex
how does affinity work?
Depending on the number and strength of the non covalent interactions, the binding between two molecules may be tight or loose, and consequently be either long-lasting or short lived.
The greater the number of interactions, the better the specificity.
The higher the affinity of two molecules, the better the ‘molecular fit’ between them, the more non covalent interactions can form and the more tightly they bind (usually - some exceptions to this in future years)
why do proteins bind to other proteins?
to form higher order structures, or protein complexes.
a - shows the same protein dimerising - in this case it’s a protein in the electron transport chain called Cytochrome C. The individual components of the protein bind one another by a particular surface of the protein.
b - shows an example of a protein ligand (interleukin-2) binding it’s specific receptor, Interleukin-2 receptor.
Binding events can profoundly influence protein function
what is molecular complementarily followed by usually?
followed by an element of induced-fit
There is a close complementarity between the structure and shape of a substrate and its binding position in the active site. This led Emil Fischer to the ‘lock and key’ analogy for the fit between a substrate and an enzyme. Later data established that enzymes permit some (small) latitude in the structure and shape of a substrate, leading to the ‘induced-fit’ concept (Koshland).
explain the schematic of a protein kinase called cAMP-dependent protein kinase A.
There are two Regulatory Subunits (R) and two Catalytic Subunits (C).
R - has binding sites for cAMP.
C - contains a kinase activity
R and C associate into a R2C2 complex, held together tightly by non-covalent interactions between the different subunits.
In the R2C2 form, the kinase activity of the C subunit is OFF.
what happens when cAMP levels in the cell rises?
cAMP is tightly bound by the R subunits.
This results in a conformation change (shape change) of R subunits, and so weakens the association between R2 and C subunits.
C subunits fall off and the catalytic activity of the C subunit is turned on - the kinase can then phosphorylate its targets.
this produces the same 4 x cAMP molecules that were added to the reaction
what key concepts does the example demonstrate?
Proteins can bind small ‘ligands’ (like cAMP) and proteins can also bind to other proteins.
Binding of a ligand (or another protein) can induce a change in protein conformation (shape).
This can be associated with a change in activity.
Binding can be quantified and measured
what is affinity?
Biologically, we define this by the binding dissociation constant, Kd.
Consider a protein P that interacts with Ligand L
P + L (front and back arrows) PL
front = Kon back = Koff
The dissociation constant Kd is calculated from the concentrations of the three components when they are at equilibrium
Kd = [P].[L] / [PL]
Kd = koff/kon
what does it mean for a low Kd
The lower the Kd, the lower the concentration of L is needed to bind half of P.
Hence, the lower the Kd, the tighter the binding and hence the higher the affinity.
A large biomolecule like a protein can have multiple binding surfaces and exhibit very tight binding.
how do we measure binding?
One of the key functions of proteins is they bind (each other, drugs, ligands, etc.)
How can we measure Kd?
Two general options are available to measure affinities:
In an equilibrium experiment, one determines the extent of the reaction as a function of the concentration of one of the reactants. Analysis of these data gives the equilibrium constant.
In a kinetic experiment, one determines the rates of the forward and reverse reactions as a function of the concentration of one of the reactants. Analysis of these data gives the rate constants for the forward and reverse reactions. The ratio of these rate constants gives the equilibrium constant.
example of equilibrium binding experiment…
Consider a labelled ‘ligand’…e.g. [3H]-cAMP ( ) binding to purified Regulatory subunit ( ) of cAMP-dependent protein kinase A.
In this kind of experiment,
a dialysis chamber is used. There are two sides of the chamber, separated by a semi-permeable membrane. The membrane has holes that allow free diffusion of small molecules like cAMP.
cAMP is added to one side of the chamber at a known concentration.
After a few hours, when equilibrium is reached, the [cAMP] on each side of the semi-permeable membrane will be equal.
Suppose we replicate the experiment, but this time we also add R-subunits. These protein subunits are too big to pass through the semi-permeable membrane….but cAMP can still move….unless its bound to R-subunit
The free [cAMP] concentration reached at equilibrium will reflect the fact that some of the cAMP has bound the protein…
How much cAMP bound at any given concentration of cAMP reflects the affinity of the R-subunit for cAMP. the left side has [FREE ligand] + [BOUND ligand] and the right Side has only a free ligand
Because the cAMP is radioactive, you can measure these concentrations accurately. Vary [cAMP] and determine [Bound] and [Free] at
constant protein concentration
what key concepts are explained through this example?
Proteins can bind small ‘ligands’ and also bind to other proteins.
Binding of a ligand or another protein can induce a change in protein conformation (shape).
This can be associated with a change in activity.
These kinds of changes can be regulated. Binding can be turned on or off - we will see examples of this in the signalling lectures later in this course.
Binding can be quantified.
But…we need structural information to really understand this. How can we get it?
