advances-in-protein-chemistry

protein demonstrate the lowest energy (ground state) conformation and function that rely on higher energy conformations of the enzyme
and its substrates [19].
Protein folding is normally argued with the help of energy landscape, showing folding funnel, which portrays thermodynamic
development through structures or sub-states leading towards the ground-state conformation of the protein. Multiple folding paths
consist of various combinations of sub-states and all available protein conformations correspond to the conformation ensemble.
Various computational and spectroscopic methodologies have been developed to observe multiple aspects of protein dynamics. X-ray
crystallographic analysis yields some dynamic information. Temperature factor is susceptible to the mean square displacements of atoms
because of thermal motions and can be acquired for nearly all heavy atoms. On the other hand, it does not give details on the time scale of
thermal motions and difficulty associated with crystal lattice contacts, refinement and static disorder procedures make their explanation
relatively difficult [19].
X-Ray and neutron scattering
Neutron scattering and X-ray give insights on dynamics of protein. X-ray scattering usually illustrates global/large changes in size
of protein and shape in a time-resolved manner and neutron scattering illustrates on amplitudes and time scales (10-12 to 10 8 s) for
hydrogen atom according to its location in protein structures.
Fluorescence technique
It is an important technique in visualizing the dynamics of protein both in single molecule and in the ensemble. Single molecule
experimentations are demonstrating to be helpful, leading towards the understanding of the motions of individual molecules of protein
as well as demonstrate how to form through translation into an ensemble signal. Computer program/simulations provide as theoretical
ground for predicting and analyzing protein motion, processing inputs from variety of experimental techniques, and probing dynamic
information beyond what can be evaluated practically. In silico techniques help to understand dynamic data that cannot be provided by
experimental methodology. Several computational programs have been developed to obtain the data/information on protein dynamics
and structural changes. Among the computational programs the Monte Carlo (MC) and Molecular Dynamics techniques are most
famous one. The precision of these approaches rely on the protocols used and on the length of simulation. By using the realistic force
fields, at most a few nanoseconds (10 -9 sec) for a small protein in aqueous environment could be reproduced within acceptable computer
time. More than 90% the internal protein motions are due to free and small fluctuations of atoms. Such internal motions show local effect
and are taken as unimportant for the analysis of protein function. The Concerted Motions (motions of interest) are those that extend over
a large number of atoms and responsible for the large structural alteration in the protein. NMR is an effective experimental technique
used for the study of protein dynamics. Time scale available to NMR ranges from 10-12 to 105 sec, and covers all the relevant dynamic
motions in proteins.
Hydrogen exchange mass spectrometry
Hydrogen exchange together with mass spectrometry (MS) is used for the study of protein dynamics. The specific protein functions
depends upon the dynamics of protein such as protein translocation from one side to the other while binding with ligand other
macromolecules or conformational changes during enzyme activation. Hydrogen exchange method is made possible because reactive
hydrogen in protein is exchanged with deuterium atoms when protein is placed in heavy water solution and the protein mass is measured
with the help of high-resolution mass spectrometry. The position of deuterium assimilation is found by examining deuterium merging in
peptic segments that are prepared after the labeling reaction.
Positions of hydrogen at peptide amide linkages (backbone amide hydrogen) are substituted with deuterium within 1-10sec when
peptides are incubated in heavy water. In case of folded proteins some backbone amide hydrogen is replaced quickly and other replace
after the periods of months. Rates of the majority slowly exchanging hydrogen may be reduced (Englander and Kallenbach, 1984).
Fourier Transform Infrared (FTIR) Spectroscopy
Infrared (IR) spectroscopy is one of the well recognized experimental techniques for the investigation of secondary structure of
polypeptides as well as proteins. IR spectrum can be achieved for proteins in a large range of environments with a small amount of sample.
IR gives information on protein dynamics and structural stability. Fourier transform infrared (FTIR) spectroscopy is well-established and
valuable instrument for the inspection of protein conformation in water (H2O) based solution, as well as in deuterated (D 2
O) forms and
dried states, ensuing in the scrutinizing the protein secondary structure and protein dynamics. Moreover, FTIR spectroscopy assists in
measuring the wavelength and intensity of the absorption of infrared (IR) radiation by a sample. The IR spectral data of sample (especially
high polymers) are generally interpreted in terms of the vibrations of a structural repeating unit. The repeating units in polypeptide and
protein provide nine characteristic IR absorption bands (amide A, B, and I to VII). Furthermore, the amide I and II bands are taken as
two most important vibrational bands of the protein back-bone [27].
Infrared spectroscopy is used for the diagnosis of cancer, due to the sensitivity of the technique to alterations in biochemistry of
biological systems which escort pathological stages. Infrared radiation (IR) is absorbed by biological system (cells, tissues and fluids) to
promote vibration of the covalent bonds of molecules within the sample. It has been observed that the proportional analysis between
FTIR spectra and histopathological investigations of normal and tumor breast cells showed that FTIR spectroscopy is a trustworthy
method for tumor diagnosis [28].
Circular Dichroism (CD)
Proteins that have been purified from tissues or obtained using recombinant techniques are studied by Circular Dichroism (CD)
which is a remarkable instrument for rapid determination of the secondary structure and folding characteristics of proteins. It is well
established that rapid categorization of new proteins is of great significance for the fields of proteomics and structural genomics. With the
help of CD multiple samples containing 20µg or less of proteins in physiological buffers could be measured in a few hours. On the other
hand, it does not give the residue specific data/information that can be obtained by NMR or X-ray crystallography. Principally, it works
on the unequal absorption of left-handed and right-handed circularly polarized light.
Raman spectroscopy
The structure of unfolded polypeptides is studied by Raman spectroscopy. Raman spectroscopy has the benefit of several essential
OMICS Group eBooks
012