BIOL 200 Lecture Notes - Histidine, Conformational Change, Amphoterism

18 views8 pages
6 Apr 2012

For unlimited access to Class Notes, a Class+ subscription is required.

Naveen Sooknanan McGill Fall 2011
Protein Structure, Function and Separation Strategies:
As we have seen over throughout this course, proteins have a wide variety of functions which
depend on their structure.
Some specific domains of proteins can carry out independent functions
o An example is the DNA binding domain found in transcriptional
activators such as homeodomain proteins
Other proteins have structures which confer very unusual and peculiar
o An example if the green fluorescent protein
(GFP) whose barrel-like structure allows
fluorescence when shone with specific
frequencies of light
o GFP can be fused into a desires protein by
placing the GFP gene directly downstream
of the protein’s gene
This causes the mRNA to have the GFP mRNA causing the
translation of a fusion protein by the ribosome
During transcription, RNA polymerase II (or whichever is
used) skips the stop site of the desired proteins gene and performs “read-
through” right to the end of the GFP gene
X-ray diffraction and crystallography are two techniques widely used to accurately determine the
structure of proteins
High concentrations of a purifies proteins, perhaps made from bacterial
overexpression, tend to form crystal lattice structures
o This structure corresponds to the protein’s lowest energy state
These crystals are bombarded with high energy beams, such as X-rays
which reflect and scatter once they hit the protein
o These beams are supplied by a piece of machinery called an
electron collider
o These radioactive scatter patterns are unique in every proteins and
are directly relates to the protein’s structure
Complex programs are able to take this scatter data and determine the
function of the protein through prediction of electron density
This process is analogous to determining the shape of a rock from examining its ripple
pattern in water
The product of this technique is an electron density map which can be analyzed in order to
determine structural aspects of the protein
Through these maps, skeletons and models can be built by placing
corresponding amino acids into place which correspond to various areas
on the map
First, the amino acid chain is produces and then any secondary, tertiary
and quaternary structure is determined through hydrophobic, hydrophilic
and other types of interactions
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 8 pages and 3 million more documents.

Already have an account? Log in
Naveen Sooknanan McGill Fall 2011
o This has been useful in molecular biology because we can manipulate proteins by
slightly altering their shape and seeing what this does to the function
Looking at the amino acid sequence, however, can only give scientists a crude
approximation of what the actual protein will look like
Thus, looking at the amino acid sequence alone can be deceptive and
lead to false assumptions with respect to the function
By determining the function of the entire protein through a method
such as X-ray crystallography, it is possible to get a better
understanding of the function of the protein as a whole
o For example, a characteristic helix structure in membrane
proteins (such as ion channels) give these proteins the function
of embedding themselves within the plasma membrane
As we have seen time and time again, protein can have catalytic activities in the body. These are
known as enzymes
The reaction between two (or more) substrates for the formation of
a product (or products) always requires an input of energy to enter
a transition state
o This is known as the activation energy of this reaction
Once the activation energy barrier is reached, the reaction proceeds
downhill (releasing energy) until it reaches product formation
o The product is either a higher or lower energy state than the product, classifying
the reaction as endothermic (using energy) or exothermic (releasing energy)
The purpose of an enzyme is to lower the activation energy of a
specific reaction, allowing it to happen spontaneously within the
binding sites of enzymes
o This usually happens by placing the substrates in very
close proximity to one another
o An enzyme is specific to one set of substrates and perform one specific reaction
o These substrates don’t just have to be other proteins, they can be any molecule in
the body
The enzyme gets is activity from characteristic tertiary structure which allow binding of
substrates and helps drive the reaction forwards
As we have also discussed, RNA molecules can also have catalytic activity with their
specific secondary structure
o These are called ribozymes, such as the 23S rRNA which catalyzes the peptidyl-
transferase reaction during translation
The efficiency of enzymes can be quantized (measured) and reflects specific
inherent properties of the protein
The rate of product formation the in presence of an enzyme is dependent
on the concentration of substrate
o At first, the reaction speeds up very quickly, but eventually
plateaus when the solution becomes saturated in substrate
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 8 pages and 3 million more documents.

Already have an account? Log in
Naveen Sooknanan McGill Fall 2011
The maximal speed of conversion from substrate to product is also dependent on the
concentration of enzyme
o Needless to say, more enzyme means faster product formation
o The maximal speed, however, is not very important because it does not give any
characteristic information about the protein
There is, however, a specific substrate concentration called Km,
or the Michaelis Constant, which corresponds to an enzyme
reaching half of its maximal speed
o This value is significant because it is INDEPENDENT
of substrate concentration
o Km therefore, is an intrinsic property of a protein and is unique to the enzyme
being tested
Protein can also bind to other molecules in a non-catalytic manner. These specific interactions
happen between a protein and a binding entity known as a ligand.
Some common ligands include growth hormones, steroid hormones and cytokines, which
all bind to specific protein receptors
Another very important protein-protein interaction is between an
antibody and an antigen; the basis of the immune system and many
protein isolation techniques as we will see soon
GTP-binding proteins are also considered ligand (GTP) binding
Ligand binding can act as allosteric switches because they can considerably change the
conformation of the protein, either activating or inactivating it
The strength of a protein-protein of protein-ligand interaction is given by the dissociation
constant, Kd
o Kd is mathematically defined as the product of the
concentrations of the two separate proteins (or ligand)
over the concentrations of complexed proteins or protein/ligands in the solution
o A high Kd represents a high amount of dissociation or a weak interaction between
the protein and ligands
o A low Kd represents a low amount of dissociation or a strong interaction
Many proteins need calcium in order to function properly. These proteins are mediated by a
calcium binding protein called Calmodulin
Calmodulin in its bare form has a radically different structure
than its calcium bound state
o Calmodulin is made of four domains: EF1, EF2, EF3 and
EF4 which can each bind one calcium atom
Binding of calcium to calmodulin causes the protein to circularize.
This allosteric conformational change activates the calmodulin
o Activated calmodulin is able to recognize specific target
peptides and cause conformational changes which are
calcium dependent
Proteins can also act as switches in the cell, turning on an off various cellular processes when
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 8 pages and 3 million more documents.

Already have an account? Log in

Get access

$10 USD/m
Billed $120 USD annually
Homework Help
Class Notes
Textbook Notes
40 Verified Answers
Study Guides
1 Booster Class
$8 USD/m
Billed $96 USD annually
Homework Help
Class Notes
Textbook Notes
30 Verified Answers
Study Guides
1 Booster Class