Lecture 1
The dimensions of the proteic macromolecules
The size of a protein can vary from a dozen to hundreds of Å, depending on
the number of domains and amino acids in each domain. In Figure 1 is
reproduced the hemoglobin molecule in comparison with other four
molecules, two of which (the heme group and the lysine) are part of
hemoglobin itself. The hemoglobin can be approximated to a sphere with a
diameter of about 60 Å and can be considered a protein of average size. Of
the four molecules, the water is the smaller one and can be approximated to a
sphere with a radius of 1.4 Å. This sphere is the probe that is scrolled on the
surfaces of proteins with known 3D structure, to define the surface accessible
to the solvent.
Figura 1. Dimension of several molecules
in comparison with hemoglobin
Structural constrains
WE will now take in consideration the structural elements of a proteic
macromolecule. The 20 amino acids have a central Cα carbon to which is
attached a hydrogen atom, an amino group and a carboxylic acid group. The
fourth chemical bond attached to the carbon atom is the side chain of the
amino acid . In all amino acids except glycine, the fourth chemical groups
linked to Cα are different and therefore all the amino acids except glycine are
1
chiral molecules. The components of protein biosynthesis have evolved to use
one of the two chiral forms, and, specifically, the form L. This form can be
distinguished by placing the amino acid as in Figure 2, where the Cα-H bond
is out of the plan of the paper, the carboxyl is located on the left, the side chain
at the center and the amine group on the right, so that you can read C_O_R_N
from left to right..
Figura 2. An L aminoacid seen along the H-Cα
bond
The preference for L aminoacids is one of the structural constraints that
impose a limit on the type of 3D structures that a protein can reach. In fact the
preferential structures are selected primarily on principles based on energy and
structural stability. Moreover, the structure is well correlated with the function,
or rather, there is a clear correlation between dynamics structure and function.
The protein molecules in fact are not static, but flexible to make the
conformational changes necessary to function and carry out a molecular
communication between different domains or molecules.
The various structural constraints present in a macromolecule will be briefly
analyzed. The peptide bond that occurs between two amino acids after
removal of a molecule of water is shown in Figure 3.
Figura 3. Linear representation
of a polypeptide
2
The first structural constraint comes from the peptide bond that is planar and
so it is possible to depict the protein through a series of plans from which
emerge the various side chains. So the first constraint is determined by the fact
that the peptide bond is forcing a number of atoms to be on the same plan. The
reason for the flatness of the peptide bond is purely energetic. As shown in
Figure 4, the atoms could be considered to combine to form a double bond
between C = O, giving rise to a free rotation around C-N bond, or to form a
double bond between C = N, however, causing a strong relocation of charge.
Figura 4. Different electronic
distribution among the atoms
forming a peptide bond
The peptide bond originates from an electronic delocalization on the three
atoms that are constrained to be on the same plan. The delocalization lead to
an energetic stabilization, since the stability is higher when the electronic
distribution is high. In summary, a protein is the succession of a series of
plans that may orient each other and that specifically characterize the protein
itself. Another peculiarity of the peptide bond is the presence of a dipole
moment. Indeed, as shown in Figure 5, the bond is characterized by a
delocalisation of negative charge near the carbonyl group and a partial positive
charge near the amide group. The dipole moment, albeit limited, is not
negligible and affect the stabilization of a protein structure, since it is present
for each peptide bond and its contribution is particularly important for some
secondary structures.
3
Figura 5.Charge delocalization in a peptidic bond
The peptide bond, as shown in Figure 6, can be found in the trans
configuration or in cis configuration, which are identified by the dihedral
angle Ω. To define Ω is necessary to determine two geometrical plans: the first
identified by the Ciα C N atoms and the second defined by the C N Cαi + 1
atoms. Ω is the diheidral angle defined by these two plans. The cis
configuration is characterized by Ω = 0, and the trans by Ω = 180.
Figura 6. The cis e trans: configuration
for any couple of aminoacido ( top) and
fora an aminoacid couple where the
second one is a proline ( bottom)
For steric reasons the trans configuration is preferred by a factor of thousand,
except when the residue i +1 is a proline. In this case, due to the cyclic nature
of the residue, the energy difference is only a factor of 4, so the difference
between cis and trans configuration is minimal. Generally, in the protein
native state, the proline residues assume the trans configuration. However, in
certain situations it can be found in the cis conformation, for example when
the polypeptide chain has to change direction. These configurations are
stabilized by additional interactions of the tertiary structure. In the denatured
state the constraints are lower and the proline residues can be found in both the
cis and trans configuration. This implies that during the process of folding a
percentage of bonds is in the wrong location, that will require the occurrence
of a cis-trans isomerization to reach the desired configuration. The
isomerization is a slow process and often limits the rate of folding. In vivo it is
accelerated through chaperones called prolyl-isomerase. The orientation of
two peptide bonds is determined by two dihedrals angles, Φ and Ψ, which can
take only defined values. Φ is defined by the four atoms Ci N Cα Ci+1 and
4
allows rotation around the N—Cα, bond, while Ψ is defined by the four atoms
N Cα Ci+1 N and allows rotation around the Cα-C bond. The polypeptide chain
can be described by a succession of peptidic units represented by plans in
which each Cα atom except the first and the last one, belong to two
consecutive units (Fig. 7).
Figura 7. The Φ e Ψ angles in polypeptide.
All atoms belonging to these units are part of the plan. The movement around
the N—Cα bond, which sets the angle Φ will rotate all the atoms that are on
the plan with N, the rotation around the bond Cα-Ci +1, which defines the
angle Ψ, will turn all atoms that are on the plan with Ci +1 (Fig. 8).
Figura 8. The Φ and Ψ angles
Not all the values of the angles Φ and Ψ are admitted due to steric constraints
between the side chain of the amino acids and the main polypeptide chain. The
allowed Φ and Ψ angles are identified via the Ramachandran plot, where the
values of Φ and Ψ are reported each as function of the other. The
Ramachandran plot is the same for all amino acids except for glycine, proline
5
and for the residue preceding the proline, because each side chain has a carbon
atom that follows the Cα atom. Figure 9 shows the Ramachandran plot for
glycine. It is nearly symmetrical and defines four regions that can be populated
with the same probability, since the side chain of glycine is just one atom of
hydrogen.
Figura 9. Ramachandran plot for a glycine.
The situation is different if you consider an amino acid side chain with a more
extensive side chain such as alanine.
Even very extensive side chains such as phenylalanine or tryptophan have one
atom of carbon following the Cα atom, therefore all the aminoacids occupy the
same conformational space. (Fig. 10).
Figura 10. Values of the Φ and Ψ angles for
the main secondary structures
Knowledge of the Φ and Ψ angles
6
clearly defines the secondary structure of a protein that derives from the
presence of a number of amino acids in which the angles Φ and Ψ have the
same value. In particular, when Φ = -57 and Ψ = -47 for a series of repeated
amino acids, the secondary structure that they form is an α helix, whereas
when Φ = Ψ = -139 and +135, it is typical of an antiparallel β strand.
Therefore, two well-defined geometrical parameters allow you to clearly
define the secondary structure determined by a polypeptide. In the
Ramachandran plot the allowed values of the angles Φ and Ψ coincide with the
values experimentally found by X-ray diffraction of protein crystals.
By analyzing the three-dimensional protein structures deposited in the PDB
bank, (which contains the coordinates of the three-dimensional structures of
proteins) and reporting on a graph the experimental,values of the Φ and Ψ you
obtain a graph as the one in fig 11, where the experimental values (represented
by dots blacks) fall into the allowed regions.
Figura 11. Experimental values of the Φ and Ψ angles
There are cases where this rule is partially disregarded partly for functional
reasons, namely a destabilizing energy is tolerated if it confers a particular
property to the macromolecule. The regions in the Ramachandran plot for the
angles Φ and Ψ include the values for which you have a defined secondary
structure, which confers stability to a protein. In the case of proteins with a
low content of secondary structure, there is always an element of
compensation that brings stability, such as the presence of disulfide bridges.
7
The proline
The other amino acid for which there is a different Ramachandran plot is the
proline (Fig. 12).
Figura
12. Representation of a proline
In this case, the angle Φ has a fixed value of - 60-while the angle Ψ can take
two values: -55 and +145, roughly corresponding to the structure of an α helix
and of an antiparallel β-strand respectively. This means that the proline may be
in an α helix structure, although it is considered a breaker of this secondary
structure. This is due to the fact that the proline strongly influences the values
of the Φ and Ψ angles of the residue that precedes it. If this is a glycine, the
disruption is minimal because the glycine has the ability to occupy a very
lwide conformational regions. In all other cases, eg. An alanine (Fig. 13), the
residue has considerable constraints and to the α helix region is not allowed
any more.
Figura 13. Ramachandran plot for an alanine
preceding a proline
Then the residue preceding the proline is
highly restricted in the conformational
8
space that can hold, and it cannot occupy the region typical of an α helix. The
residue of proline, however, may be an initiator of α helices, since the amino
acid unable to assume the Φ and Ψ values typical of an α helix is not the
proline but the one preceding it.
Also the side chains have defined constraints, and so they are usually found in
preferential orientations. This can be understood taking into consideration a
simple molecule such as ethane (CH3-CH3). Although you can rotate in any
direction relative to the CC bond, the more stable structure is the one in which
the six atoms of hydrogen are staggered, because in this conformation, the
repulsion between them is minimized. Similarly in the case of proteins, the
side chains are characterized by the presence of carbon atoms connected by
single bonds, along which rotation can occur. Every amino acid can have
several configurations, which are determined by the minimization of the
repulsion. Again the preferred configurations are those staggered that defines
the preferred rotamers. Figure 14, for example, shows the histogram for the
preferential values of the torsion Cα-Cβ angle for the inhibitor of bovine
pancreatic trypsin.
Figura 14. Distribution of the torsion
angle Cα-Cβ for the pancreatic trypsin
inhibitor
The favourite rotamers are those that have a value of the dihedral NCαCβCγ
angle equivalent to 60 degree or its multiples that correspond to staggered
configurations thar are the most stable.
Main properties of the lateral chains
9
We recall briefly some features of the amino acids. Amino acids can be
distinguished into different classes in view of their polarity. By varying the
criterion for classification it changes the class to which the amino acids
belong. Tryptophan and arginine for example, belong to different classes if the
parameter is the polarity, but belong to the same class if the classification
criterion is the size of side chain.
The comparison of the sequence of ortologue proteins allows you to identify
the amino acids that are conserved. These amino acids are generally viable and
therefore extremely important for the protein from a structural point of view or
from a functional point of view, so their elimination would be extremely
serious for the stability (structural amino acid) or the mechanism (functional
amino acid). There are a number of amino acids that can be substituted
without disrupting the structural and functional properties of the protein. There
are others that can be substituted, but only maintaining the chemical and
physical characteristics of the side chain. Sometimes substitutions are
compensatory in the sense that we must consider at least pairs of amino acids
because a mutation is correlated with the mutation of another one.
As previously stated, to consider the general characteristics of amino acids is
not enough to assess its polarity. The polarity is an important factor, since the
proteins are in an aqueous environment, but also the size is a relevant factor,
as a large side chain can be replaced only by another of comparable size. The
variation in size affects the stability of a protein and is another important
parameter of comparison between amino acids present in the same position in
ortologue proteins.
Another factor to consider is the frequency with which a given amino acid is
represented within the proteins. Not all amino acids are represented in the
same way: there are amino acids that are not very frequent while others have a
much higher frequency, as shown in Table I.
An amino acid having a low frequency is, for example, cysteine, which has a
SH group which is highly reactive. The reactivity is useful when limited to the
active site of an enzyme, because it allows you to assign a specific function,
but a highly reactive chemical group, located in the wrong location, can give
rise to a series of reactions that can lead to the destabilization of the protein
and thus its subsequent degradation.
Tabella I. Aminoacid frequency inside proteins
10
Amino acids such as alanine or leucine have high frequency, they are inert
amino acids, are used to create structural skeletons and can be used in multiple
places as a basis on which build more reactive and functional amino acids.
Thus, the frequency of amino acids is related to their importance and their
function. Its value depends on the type of protein. If the table of frequencies is
built taking into account only a defined class of proteins such as
metalloproteins, the distribution would be different since cysteine and
histidine are the preferred ligands of metals and thus are highly represented in
this class of proteins. The table shows that the frequency of tryptophan is low.
This stems from the fact that it is a very bulky amino acid that can be
accomodated only in defined regions. It has some preferential location, for
example, in membrane proteins, it is often present between the lipid and the
aqueous phase. The tryptophan acquires a different frequency if analyzed on
protein membrane. The choice of a database of specialized proteins produces a
distorted vision or better a vision representative only of the observed class.
It may be useful to examine the characteristics of certain amino acids in order
to understand the different roles they can play in a protein.
The glycine (Fig. 15) is the amino acid that has less constraints.
Figura 15. Representation of a glycine.
Since it is the smallest amino acid, one might assume that
it can be replaced by any other amino acid. A
comparative analysis of ortologue proteins indicates that
11
the position occupied by glycine is often preserved, indicating that it plays a
crucial role in determining the structure of a protein. This is due to its low
steric hindrance and to its ability to take unusual dihedral angles values.It is
often found in turns, where the protein changes its direction of propagation.
The alanine (Fig. 16) is the most neutral aminoacid relative to the
region that can occupy. The frequency of attendance is high, the amino acid is
non-polar and can adapt itself to many situations.
Figura 16. The alanine.
Branched amino acids (Fig. 17) have more steric constraints and are suitable
to build the hydrophobic core or skeleton of a protein.
Figura 17. The branched aminoacids
In a protein structure and function are related in the sense that a given
structure is associated to a specific function. It is also true that some regions of
the protein are appointed to provide stability while others are appointed to
12
confer functionality. Therefore, the structural skeleton is provided, in general,
by amino acids with a high content of secondary structure and thefunction by
different amino acids, which often are on loops, or on regions characterized by
a relatively high mobility.
.
Figura
18.
aminoacids
The
aromatic
The aromatic amino acids are
relatively bulky, but all have a
CH2 group before their ring,
which gives a Ramachandran
plot comparable for all of them.
In Figure 18 the three aromatic
amino acids, able to absorb
electromagnetic waves in the ultraviolet region, are represented.
The aromatic rings give rise to orbitals among which transitions can occur
upon interaction with electromagnetic waves. The aromatic amino acids can
then act as endogenous optical probes to obtain structural information on the
state of the protein itself.
The interaction of a protein with the electromagnetic radiation and, in
particular, the absorption and emission spectroscopy represents a powerful
approach to obtain structural/functional informations on protein molecules,
because it allows to investigate the molecule in solution, without damaging the
molecule itself. The comparison of the characteristics of the electromagnetic
wave before and after the interaction with the macromolecule can be used to
obtain meaningful data regarding the structure of the macromolecule itself.
The length of the electromagnetic waves can range from radio frequency to Xrays, and different informations can be obtained depending on its length.
Through the spectrum of absorption or emission of aromatic amino acids, you
can get information on local structure, namely on the structure of the region in
proximity of tyrosines or tryptophans, which are responsible for the observed
signal.
The proline (Fig. 19) is an amino acid that is often found in turns and is then
used to introduce changes in the direction of propagation of the polypeptides
13
chain.
Figura 19. The proline.
Figure 20 represents polar amino acids, whose main characteristic is that of
forming hydrogen bonds through their side chain. They are important in
defining the molecular recognition and in the stabilization of the structure of
the macromolecule.
Figura 20. The polar aminoacids
The histidine is formed by an imdazolic ring separated from the Cα by a CH2
group (Fig. 21). It is often present in the active sites.
14
Figura 21. The histidine.
The ring may take three forms depending on pH: protonated,
partially deprotonated and completely deprotonated. In the protonated form,
that is positively charged, the imidazole ring has a proton on both nitrogen
atoms. The pK of the first deprotonation is 6.7. The histidine can then move
from a protonated to a partial deprotonated form at pH around the neutrality,
permitting a transfer of protons. The ring is then characterized by a second pK
at around pH 14, which leads to a completely deprotonated form that is
negatively charged.
The chemical reactivity
Some amino acids have a high chemical reactivity. Cysteine is the amino acid
showing the highest reactivity, a feature that is used in the procedures of
chemical modification of proteins. Until the'70s the study of proteins was
strongly based on the external chemical modification of the proteins
themselves. Subsequently, the introduction of recombinant DNA technology
has led to a different experimental approach, so that the amino acid, instead of
being chemically modified, is replaced by site-directed mutagenesis. Recently
the approach of site directed mutagenesis coupled with chemical modification
is widely used. For example, the cysteine is introduced in a systematic way to
perform chemical modification of its side chain to introduce a defined probe.
Indeed, cysteine, beside being a highly reactive amino acid, is the only one to
have an SH group. This implies that the cysteine may be subjected to a
selective chemical modification not involving other amino acids. Moreover,
the possibility of introducing this specific amino acid in defined positions
through recombinant DNA technology permits to obtain information on
15
known and defined regions.
Lysine (Fig. 22) is another reactive amino acid. At neutral pH, it is positively
charged, and it is often found on the outer surface of a protein, often involved
in salt bridges with negatively charged groups. At basic pH the lysine
deprotonates, the group passes from NH3+ to NH2 becoming a nucleophile.
This confers a strong reactivity and at basic pH lysine can undergo reactions
such as alkylation and acylation.
Figura 22. The lysine
Methionine (Fig. 23) is an amino acid that can react with methyl iodide (Fig.
24) to give rise to a chain with a doublelinked methyl on the sulfur atom. The
reaction may be reverted
adding thiols, to eliminate one
of the two CH3 groups.
Figura 23. The metionine.
Figura 24: Reaction of metionina with CH3I;
oxidation reaction.
Statistically in 50% of the molecules the new inserted group, and in the other
50%, the previously existing one will be removed. This procedure may be
16
useful to selectively mark a methionine. For example, you can add a CH3 with
a radioactive carbon or 13C, which is visible by NMR, and then restore the
initial conditions, having 50% of molecules labeled on CH3.
The introduction of an external probe has the inherent problem of the
perturbation, as the chemical modification,introducing a chemical group,
implies a possible structural perturbation. The introduction of a probe for local
information, can disrupt the structure of the molecule and thus ensure that the
information obtained is no longer that due to the native conformation. The
case of chemical modification of methionine is one of the few cases where it is
possible to introduce a probe without causing perturbation.
Methionine is also susceptible to oxidation, which can occur in two steps: the
first is reversible, while the second is irreversible and causes permanent
damage to the protein itself, which becomes more easily degradable (Fig. 24).
The most reactive aminoacid is the cysteine (Fig. 25).
Reactivity is due to the presence of the thiol group that is
reactive in the deprotonated form. The reactivity is then high
for pH greater than 8 when the group will be in the
deprotonated form, although the pK of this group can be
strongly influenced by its environment.
Figura 25. The cysteine
At partial alkaline pH cysteine can react with double bonds, for instance it can
covalently react with N-etilmaleimmide (Fig. 26).
Figura 26. Reaction of cysteine with Netilmaleimmide.
The maleimmide is a group that is used
very often for the reaction of cysteine and
17
it is used to insert different reporter groups. The frequency of the presence of
cysteine in a protein is very low, as in about one hundred amino acids it is
usually present only one cysteine. In order to introduce a probe in a specific
position it is necessary to have a single cysteine. This can be achieved firstly
removing all the cysteines present in the native form by mutagenesis (the
serine is the replacing aminoacid generally better tolerated) and checking that
the functional properties are not perturbed. Then it is possible to introduce a
single cysteine in defined regions and make a systematic study through the
introduction of a selective probe. An useful chemical modification is the one
with the mercury benzoic acid, which allows the introduction of an aromatic
ring with defined pH dependent absorption properties since its extinction
coefficient ε varies with pH (Fig. 27).
Figura 27. Reazione della cisteina con acido mercuro benzoico.
1 The extinction coefficient ε is a characteristic property of a chromophore absorption
at a defined wavelength, for example, ε255 mean molar extinction coefficient at 255
nanometers. A macromolecule with an internal chromophore is able to absorb light
with a wavelength in response to radiation. In the specific case in which in the cuvette
there is a protein molecule modified with mercury benzoic acid, if the sample is
irradiated with a wave of intensity I0 and wavelength of 500 nm, no changes will be
obseved and the same intensity I0 will be observed before entering and after leaving
the sample. When the sample is irradiated by a wavelenght of 255 nanometers, the
intensity of the outgoing radiation I will be lower than the incoming one I0, because
part of it will be absorbed by the sample. The logarithm of the ratio I0/I correspond
to the absorbance. The absorbance is related to the extinction coefficient ε via the
18
relation A = εcl, where c is the concentration of the sample and l is the length of the
cuvette. The c is expressed in molar and l in cm. As the absorbance is dimensionless,
ε has the dimension of M-1 cm-1. For a very long cuvette, the absorbance will be
greater, compared to a small cuvette. In the case of mercury benzoic acid, the
extinction coefficient ε is a few thousand, a relatively high value. High extinction
coefficients indicate that the transitions are allowed and, in this case are more allowed
at neutral pH than at acidic pH.
19
Two cysteines may react one with another and create what is called a disulfide
bridge (Fig. 28), obtained by oxidation of two thiolic groups. The reaction is
reversible, as in the presence of a reducing agent, the disulfide bridge is
reduced in two cysteine. The disulfide bridge confers stability and can be
engineered within a protein to increase stability, but to be formed, defined
geometric conditions must be respected and the system must be in a partially
oxidizing environment.
Figura 28.A disulfide bridge
Figure 29 shows another thiolic groups reagent, the ditiobisnitrobenzoic acid
(DTNB), often used for determining the number of cysteines present in a
protein.
Figura 29. Reaction of a cysteine with DitiobisNitroBenzoic acid (DTNB).
The DTNB is able to react by generating a mixed disulfide in which a sulfur
atom is provided by the protein and one by the reagent. Its reactivity with
cysteine is high. The DTNB is used to determine the presence and number of
cysteines in a protein The quantification is made measuring the absorption,
because the anion that is released is capable of absorbing electromagnetic
wave length at 412 nm with an ε of 13,600 M-1cm-1, a value which makes
possible the quantification even of a low concentrated protein solution. The
20
mixed disulfide can be reduced through the addition of a reducing agent, such
as dithiothreitol (DTT), which releases a new anion with similar absorption
characteristics. Then, after the titration of the cysteine of the protein by
reaction with DTNB, you can check the number again, reducing the protein
with DTT and showing that the absorbance that emerges is identical to that
obtained by titration with DTNB.
The reactivity, or its speed, is a reporter of the microenvironment, as it gives
information on the availability of the side chain to react. The factors that can
modulate the reactivity of an amino acid are shown in Figure 30.
Figura 30. Factors modulating the
reactivity rate of an aminoacid.
The pH is an important issue, for example lysine and cysteine, require a
relatively alkaline pH to be reactive. The deprotonation of a group depends on
its pK, determined by the microenvironment, i.e. by the distribution of
chemical groups around the single amino acid . This suggests that following
the reactivity as a function of pH may provide information on the pK of the
group and its chemical surrounding.
The three elements that modulate the pK of a chemical group are:
•-electrostatic factors: the presence around a position of positive or negative
changes strongly influente the pK of the group ;
•-solvation: the accessibility of the group to the solvent and its dielectric
constant modulate the capacity of the group to react with external agents,
•-hydrogen bonds: a group engaged in hydrogen bonds with other molecular
partners, has more difficulty in reacting with an external agent
In addition, an amino acid, to be reactive, must be accessible to the reagent
and thus steric factors are certainly important.
21
The chemical modification allows the 'insertion of exogenous probes, which
can introduce perturbation as well. When possible, it is preferable to use
intrinsic probes, in order to prevent the introduction of exogenous molecules.
Spectroscopic properies of aromatic aminoacids
The aromatic amino acids, which have a characteristic absorption and
fluorescence spectrum (Fig. 31), are an excellent example of endogenous
probes.
Figura 31. Absorption spectrum of a tryptophan and a tyrosine.
The absorption at 280 nm, the wavelength at which the aromatic residues
absorb, is widely used to quantify the concentration of a protein. Each protein
has a defined extinction coefficient at this wavelength, depending on the
number of aromatic hydrocarbons and their chemical surrounding. The
environment is indeed able to modulate the intrinsic extinction coefficient of
each aromatic group. The two amino acids that have the highest extinction
coefficient are the tryptophan and tyrosine, as reported in Table II which
shows the coefficients corresponding to the absorption maximum for
tryptophan, tyrosine and phenylalanine.
For both tyrosine and tryptophan the maximum is centered at around 280 nm
and the intensità is about 4 times higher in tryptophan (Table II).
Tabella II. Parametri spettroscopici degli aminoacidi aromatici.
22
In figura 32 the absorption spectra of the aromatic aminoacids are reported
Figura 32. Absorption spectra of the aromatic residues.
The extinction coefficient of phenylalanine is 200 M-1cm-1, an order of
magnitude lower than that of tyrosine and tryptophan and therefore negligible,
but the shape is very articulate and can contribute to the final shape of the
absorption spectrum. In the case of tyrosine, the absorption spectrum depends
on the pH value, since the hydroxyl group looses the proton at high pH values.
The shape of the spectrum changes strongly with pH and in figure 32 it can be
seen that atalkaline pH it has a maximum at 295 nm, while at acid / neutral
pH, at this wavelength, there is not absorption. At alkaline pH the band at 295
nm is characterized by an extinction coefficient ε of 2500 M-1cm-1.
An example of the information
coming from the absorption
spectrum of a protein is reported
in Figure 33, where the change
in the absorption spectrum as a
function of pH in the UV region
of the ribonuclease protein is
reproduced
Figura 33. Absorption at 295 nm in
ribonuclease as a function of pH.
23
In detail: the absorption spectrum was followed at a wavelength of 295 nm, ie
the wavelength where the tyrosine absorbs at alkaline but not at neutral pH.
This implies that the absorption spectrum at neutral pH of a protein, not
having tryptophans, is completely silent at 295 nm. The figure shows the value
of the extinction coefficient at 295 nm as a function of pH. The value of the
extinction coefficient is equal to 2500 M-1cm-1 when a single tyrosine
iscompletely deprotonated, equal to 5000 M-1cm-1 when two tyrosines are
fully deprotonated and so on. At pH values around 11/11.5 the observed
extinction coefficient is 7500 M-1cm-1 . This result indicates that, starting
from pH 6 and reaching pH 11, three tyrosine are titrated and that for this
protein there are three accessible tyrosine that are titratable within the pH
considered. So in the absence of knowledge on the protein structure,
measuring the absorption as a function of pH, it is possible to demonstrate the
presence of three tyrosine with a pK around 10 and then TITRATABLE in pH
ranging from 6 to 11 / 11.5. This is confirmed by the fact that the titration is
completely reversible. Indeed, lowering the pH from pH 11 to 6, we obtain a
spectrum identical to that obtained by increasing from 6 to 11. This suggests
that these changes in pH did not irreversibly disturbed in the structure of the
protein, but resulted only in a reversible reactions of protoation and
deprotonation. When the pH is further increased, from pH 11 to pH 13, the
absorbance (and therefore the coefficient of extinction) continues to increase
and, at pH 13, the extinction coefficient is 15,000 M-1cm-1. This means that
three other tyrosines were titrated, as the extinction coefficient has been
doubled from 7500 M-1cm-1 at 15000 M-1cm-1. This result implies the
presence of six tyrosine: three reveribly titratable within pH 11, the other three
deprotonable only at higher pH and thus probably only irreversibly. In fact,
bringing the pH 13 to neutral pH, the absorption spectrum is not identical to
that obtained increasing the pH. This means that the initial condition is
reached through a different path and that the phenomenon is not reversible. To
deprotonatethe other three tyrosine was necessary a large conformational
change that partially denature the protein itself. The three tyrosines are
inaccessible to the solvent in the native state, so that their accessibility can be
achieved only by protein denaturation.
This example has shown that the acquisition of the absorption spectrum in the
UV region as a function of pH can give information on the quantity and
accessibility of tyrosine residues within a protein. In this case the first three
tyrosine are accessible to solvent: hey are three amino acids that have the side
chain exposed to solvent and therefore characterized by a pK typica of an
unscreened tyrosine. By contrast, the other three tyrosines are in an
environment masked by other residues and thus can deprotonate only after d
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proteindenaturation. In Table II, in addition to the absorbance maximum, also
the fluorescence maximum of the aromatic amino acids are reported
Fluorescence spectroscopy is an emission spectroscopy that, like the
absorption spectroscopy can provide information on the local structure of a
protein macromolecule. The phenomenon of fluorescence is due to the
radiative emission of the absorbed radiation. When radiation is absorbed, the
absorbent system is brought from the ground state to an excited and the system
can return to its initial state dispersing the absorbed energy. The energy can be
dissipated in various ways, the most usual one being heat dissipation. When
the system returns to the ground state emitting a radiation the phenomenon is
called fluorescence (Fig. 34).
Figura 34. Sheme of the relaxation processe from an excited state.
The fluorescence is a technique much more powerful than optical absorption
and allows the execution of sophisticated experiments that can give
nformation both on the structural and dynamical properties of a protein. A
feature that can be exploited to provide information regarding the position of
fluorescent probes is the dependence of the wavelength of emission from the
accessibility of the probe to solvent. Figure 35 describes the dependence of the
wavelength of emission of the indole group to the composition variation of
two different solvents: cyclohexane and ethanol.
Figura 35. Solvent effect on the
emission wavelength of an indolic
group
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Initially, the indole is dissolved in cyclohexane and has an emission spectrum
centered at 300 nm. Increasing the percentage of ethanol, which is a relatively
polar solvent, the emission spectrum shifts to longer wavelengths centered at
350 nm when dissolved in pure ethanol. This result indicates that the indole
feels the solvent and that the energy levels are rearranged, reaching different
energies depending on the solvent. In ethanol the emission wavelength
increases so that the energy difference between the two levelsinvolved in the
emission process decreases. The emission spectrum of tryptophan depends on
its location in a hydrophobic or hydrophilic environment. In this experiment
the ethanol represents the hydrophilic environment and cyclohexane the
hydrophobic one. Because the wavelength of the emission spectrum is strongly
influenced by the environment, the spectrum provides direct information on
the chemical environment of the chromophore. Figure 36 shows the emission
spectrum of a protein that in the native state has a tryptophan that is located
within the hydrophobic core, completely shielded from the solvent, compared
to the spectrum of the same protein after denaturation when the tryptophan is
accessible to solvent.
Figura 36. Fluorescente spectrum of a protein in
the native and denaturated state
Table III shows the emission maximum of proteins that contain a single
tryptophan. The maximum varies from protein to protein and indicates that its
solvent accessibility. In azurin the tryptophan is completely shielded and
inaccessible to solvent while the tryptophan in glucagon is located on the
surface of the protein. In the latter case, the tryptophan is not a good probe to
monitor the process of denaturation of the protein itself.
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Tabella III.Emission maximum of proteins containing a singe tryptophan
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