advances-in-protein-chemistry

egion) for estimating fraction or percentage of different secondary structural elements in proteins [69,71-73]. The data
obtained can be analyzed through different algorithms that are based on the CD spectra of proteins of various fold types
that have already been solved by X-ray crystallography. Some free programs that are widely used include DICHROWEB
[74,75] and K2D2 [76].
Tertiary structure information for proteins using CD spectroscopy is ascertained by analyzing CD spectra obtained
between wavelengths 260-320nm [68]. Spectrum in this region is a contribution of aromatic amino acids. The characteristics
of near UV CD spectrum of a protein like shape and magnitude will depend on many factors like type and number of
aromatic amino acids, mobility and nature of their environment and their spatial distribution within the protein. There
hasn’t been much advancement in use of near UV CD spectra to gain significant structural insights, nevertheless in some
cases like bovine ribonuclease [77] and human carbonic anhydrase II [78] assigning features of spectrum to particular
amino acids by sequential removal of these residues by site-directed mutagenesis has been accomplished. Near UV CD
spectrum can also provide important evidence for existence of ‘‘molten globule’’ states in proteins, which are characterized
among other things by very weak near UV CD signals [79,80].
Ligand binding induced conformational changes in proteins are an essential part of their mechanism of action and
biological activity and can be visualized through CD spectroscopy [81-83]. These changes can be monitored both as a
function of ligand concentration and function of time (in time-resolved CD studies) [73]. Former leads to information
about effective optimal ligand concentration while as latter provides information about response time to generate effect of
ligand binding.
Structural integrity and folding properties of proteins can also be studied using CD spectroscopy. Contribution of
different amino acids towards protein structure can be identified by combination of site-directed mutagenesis and CD
spectroscopy. Coupling these experiments to chemical and thermal denaturation experiments can lead to information about
relative contribution of amino acids. Mechanism of protein folding using CD spectroscopy has been studied by measuring
rate of acquisition of secondary and tertiary structure of a denatured protein. Information like this can be ascertained by
studying folding events at millisecond or sub millisecond scales using continuous or stopped-flow CD methods [84-87].
Fluorescence Spectroscopy
Absorption and CD spectroscopy involve excitation from ground state to a higher energy state with expected return to
ground state. This return to ground state is accompanied by release of the absorbed energy in a form such as heat that is not
further informative. However in some cases absorbed energy is released in other forms that can lead to further information
about characteristics of material emitting absorbed energy. Fluorescence is one such mechanism usually classified under
‘emission spectroscopy’ which involves transitions of electrons between different vibrational states in ground and excited
states, the return to ground state being accompanied by release of photons. The ratio of the number of photons emitted to
the number of photons absorbed known as fluorescence quantum yield gives information about efficiency of fluorescence
process [2,88]. Intrinsic fluorescence in proteins is a collective contribution from its aromatic amino acids tryptophan,
tyrosine and phenylalanine. They differ in absorption (excitation) and emission wavelengths, fluorescence life times and
their fluorescence quantum yields [Table 2]. Due to differences in excitation and emission wavelengths there is resonance
energy transfer from phenylalanine to tyrosine and from tyrosine to tryptophan, as a result of which the fluorescence
spectrum of a protein mostly resembles that of tryptophan [88].
Fluorophore Excitation Wavelength Emission Wavelength Lifetime (10 -9 sec) Quantum yield
Tryptophan 280nm 348nm 2.6 0.2
Tyrosine 274nm 303nm 3.6 0.14
Phenylalanine 257nm 282nm 6.4 0.04
Table 2: Fluorophores in proteins.
Application: Fluorescence spectroscopy has been extensively exploited in protein characterization. Proteins have
intrinsic fluorophores which form readily available centers that can provide information about protein structure. Further
quantifiable changes in characteristics of these fluorophores can be monitored for changes in protein conformation. In
addition proteins and other protein interacting molecules like nucleotides and DNA can be covalently linked to a small
fluorescent molecule which makes them very useful in studying macromolecular interactions and enzymatic reactions.
Some applications have been elaborated below:
Intrinsic fluorescence spectrum of a protein in native conformation is characteristic of that particular protein,
although measure of intensity of fluorescence is not much informative. The value of quantum yield is dependent on other
environmental factors like temperature, solvent and presence of other chemical species that might contribute to increase or
decrease of fluorescence intensity [2,88,89]. Thus information about presence of tryptophan in a specific environment can
be deduced from its emission maxima and to some extent intensity also. A solvent exposed tryptophan having interactions
with surrounding residues will have a red-shifted emission maximum as opposed to buried tryptophan residue which will
have a blue-shifted maximum [2,88,89]. Of particular interest are those tryptophan residues which are in proximity to a
ligand binding site of the protein, because they can lead to information about protein topography [89-92]. Quenchers like
acrylamide and heavy metal derivatives are used as probe to gain information about molecular make up especially nature
of charged residues around active sites [92,93]. Similarly quenchers serve as means to monitor the extent of conformational
changes as a result of ligand binding.
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