advances-in-protein-chemistry

A=ε*c*l (13)
where constant c in equation 13 is the molar concentration of protein, A is absorbance A 280
and l is path length in cm and
ε, the extinction coefficient is a protein characteristic quantifying the total contribution of different chromophores present
in the protein (2). It is estimated as
ε 280 (M -1 cm -1 )=#Trp*5500+#Tyr*1490+#Cys*125 (14)
Concentration in mg/ml can be estimated as molar concentration divided by molecular weight of protein.
Conformational changes in proteins due to structural perturbations or ligand binding can be monitored using
absorption spectroscopy by studying changes in chromophore and consequently in spectroscopic properties of protein
[63-65]. These changes in chromophore can arise either due to change in the chromophore environment as a consequence
of conformational changes or can come from direct chemical interactions of binding molecule with chromophore. These
differences can be quantitatively studied by measuring the change in absorbance (ΔA) or what is called ‘difference spectrum’.
Difference spectra are depicted by plotting differences in absorbance of the protein molecule under study in two conditions;
native condition and one in which the difference is intended to study.
In addition to utilization of intrinsic spectral properties in studying proteins, external spectroscopic probes can also be
used to ascertain protein characteristics. An example of one such probe is 5, 5’ dithio-bis-(2-nitrobenzoic acid) [DTNB]
which is used to measure number of free cysteine sulfhydryl groups in a protein [66,67]. DTNB undergoes a significant
change in its absorbance spectrum on forming a disulfide bond and this ability to form disulfide bonds with concomitant
change in spectral property is exploited in exploring protein composition. Similarly other probes can be used to monitor
changes in regions of protein that are in close proximity to probe binding site.
Circular Dichroism
Circular Dichroism (CD) involves studying optical properties, the principles of which are based in molecular asymmetry
or chilarity in the protein molecules. Asymmetry or chilarity in protein molecules means their structures and their mirror
images are non-superimposable and this asymmetry becomes observable when plane polarized light passes through
protein solution. Plane polarized light is composed of two circularly polarized components of equal magnitude; a counter
clockwise rotating component called left-handed or L component and clockwise rotating component called right-handed
or R component. An optically active solution like those of proteins will differently absorb this left and right circularly
polarized light [2,68-72]. This difference can be expressed by Beer-Lambert law as:
ΔA=A L
-A R
=ε L
*l*C- ε R
*l*C= Δε*l*C (15)
This difference in absorption results in production of elliptically polarized true light with angle ψ and is represented as
ellipticity, θ. Data from CD spectroscopy experiments is presented either in terms of ellipticity [θ] (degrees) or differential
absorbance ΔA. The mean residue ellipticity ([θ]mrw,λ) at wavelength λ, is given by:
[ θ ]
λ
[ θ ]
mrw,
λ
= MRW * (16)
10* d * c
where [θ] λ
is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm), and c is the concentration (mg/
ml). The Mean Residue Weight (MRW) for protein (in reality peptide bond) is calculated from MRW=M/(N-1), where M
is the molecular mass of Protein (in Da), and N is the number of amino acids; the number of peptide bonds is N-1. For
most amino acids the MRW is 110±5 Da. If molar concentration of protein is known, the molar ellipticity ([θ] molar,λ
) at
wavelength λ is given by:
100*[ θ ]
λ
[ θ ]
, λ
=
(17)
molar
m*
d
The units of mean residue ellipticity and molar ellipticity are deg cm 2 dmol -1 . When data is obtained in absorption units,
results are expressed in ‘molar differential extinction coefficient’ Δε. If the observed difference in absorbance at a certain
wavelength of a solution of concentration m in a cell of path length d (cm) is ΔA, then Δε is given by:
∆A
∆ ε =
(18)
m*
d
The units of molar differential extinction coefficient are M -1 cm -1 . There is a simple numerical relationship between [θ]
and Δε namely [θ] =3298 Δε.
mrw mrw
Application: The major center of asymmetry present in proteins is the amide group formed by interaction between
two amino acids. Further contribution to the optical activity of proteins comes from their ability to form well defined
super asymmetric secondary structural elements like α-helices, β-sheets and β-turns. These secondary structural elements
have distinctive CD spectra which in turn contribute to characteristic CD spectra of individual proteins, depending on the
relative contribution of structural elements towards overall structure of the protein. Consequently CD spectroscopy has
been used as a tool to study structural composition of proteins. CD spectroscopy has found all the more important role
in studying changes in protein structure and conformation in response to ligand binding or in response to presence of
structural perturbants like temperature and denaturing agents.
Secondary structure composition of proteins by CD spectroscopy has been studied exploiting the asymmetry generated
by peptide bond. This is accomplished by utilizing the data recorded in CD spectra of proteins below 250 nm (far UV
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