What are proteins and their importance?
Protein
is the material basis of life, an organic macromolecule, the basic organic
matter that constitutes a cell, and the main bearer of life activities. Without
protein, there would be no life. Amino acid is the basic building block of
protein.
The Basic Unit of Protein Structure - Amino Acid
There
are more than 300 kinds of amino acids in nature, and protein sequences are
composed of twenty different amino acid residues. Proteins are multimers formed
by linking different amino acids, and through correct folding into a specific
configuration, they exert the biological function of protein drugs.
Protein
structure refers to the spatial structure of protein molecules. Proteins are
mainly composed of chemical elements such as carbon, hydrogen, oxygen, and
nitrogen. All proteins are polymers formed by connecting 20 different amino
acids. After forming proteins, these amino acids are also called residues.
The
characteristics and functions of a protein sequence have always depended on the
nature, type, and quantity of the amino acid residues that make it up. Specific
positions in the amino acid sequence can be covalently bonded to chemical
groups, resulting in protein post-translational modifications, which will lead
to changes in the structure of the protein, thereby affecting the biological
activity of protein drugs.
Proteins
have the characteristics of relatively large molecular weight, complex
structure, and high heterogeneity. Therefore, their characterization is
extremely challenging, and it is very important to use appropriate methods to
characterize them.
3D
Structure of Proteins
Protein molecules are covalent polypeptide
chains formed by the condensation of amino acids end-to-end. Every natural
protein has its own unique spatial structure or three-dimensional structure.
This three-dimensional structure is usually called the conformation of the
protein, that is, the structure of the protein. The molecular structure of
proteins can be artificially divided into primary, secondary, tertiary, and
quaternary structures.
•
Primary structure: the linear sequence of amino acids that make up the
polypeptide chain of a protein.
•
Secondary structure: a stable structure formed by hydrogen bonds between C=O
and N-H groups between different amino acids, mainly α-helix and β-sheet.
•
Tertiary structure: the three-dimensional structure of a protein molecule
formed by the arrangement of multiple secondary structure elements in
three-dimensional space.
•
Quaternary structure: used to describe functional protein complex molecules
formed by the interaction between different polypeptide chains (subunits).
What
is protein characterization?
Protein
characterization analysis is to characterize the biological functions and
various properties and parameters of proteins, including protein types, protein
structures, content, protein purity, amino acid composition, molecular mass,
amino acid composition, etc. The most common way to perform protein
characterization is mass spectrometry. Mass spectrometry (mass spectrometry,
MS) analysis technology won the Nobel Prize in 2002. (In 2002, Tanaka Koichi and John B Fenn at the
University of Virginia were
awarded the Nobel Prize in Chemistry for their contribution to the soft adsorption
ionization method.)
Protein Characterization Techniques:
Mass
spectrometry:
Mass
spectrometry has good sensitivity and accuracy and can accurately determine proteins.
At present, mass spectrometry mainly determines the primary structure of
proteins, including molecular weight, amino acid sequence of peptide chains,
and the number and position of polypeptides or disulfide bonds, which occupies
an important role in the study of protein structure analysis. The mass
spectrometer consists of a sample injector, an ion source, a mass analyzer, an
ion detector, a control computer, and a data analysis system. Traditional mass
spectrometry is only used for the analysis of small molecule volatile
substances, but with the emergence of new ionization techniques, such as
matrix-assisted laser desorption ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS). The
emergence of various mass spectrometry technologies provides a new, accurate,
and fast way for protein analysis.
Mass
spectrometer technologies, such as MALDI, TOF, CFR, and hybrid quadrupole mass
analyzers, have been vigorously advanced to meet the high selectivity and high
sensitivity required for the identification and characterization of new
proteins and other biomolecules in drug discovery. [1]
The
principle of mass spectrometry protein analysis:
The
basic principle of mass spectrometry protein analysis is to convert protein
molecules into ions through an ionization source and then use the electric
field and magnetic field of the mass spectrometer to separate protein ions with
a specific mass-to-charge ratio (M/Z), and pass through the ion detector to collect
the separated ions, determine the M/Z value of the ions, and analyze and
identify unknown proteins. Usually combined with corresponding processing and
other techniques, proteins can be identified more accurately and quickly.
Circular
dichroism spectral characterization
Circular
dichroism is a special absorption spectrum. It obtains the secondary structure
of biological macromolecules by measuring the circular dichroism spectrum of
proteins and other biological macromolecules. It is simple and fast. It is
widely used in protein folding, protein conformation research, and enzyme field
of dynamics.
Far-ultraviolet
circular dichroism can be used to determine the secondary structure of
proteins, and near-ultraviolet circular dichroism can be used to detect the
tertiary structure of protein side chains.
Fourier Transform Infrared Spectroscopy Characterization
Fourier
Transform Infrared Spectrometer, abbreviated as FTIR. The Fourier transform
infrared spectrometer (FTIR) was born in the 1970s. It is composed of a light source
(carbon rod, high-pressure mercury lamp), a Michelson interferometer, a sample
chamber, a detector, a computer system, and a recording and display device. It
consists of several parts. The Fourier transform infrared spectrometer not only
has high resolution and scanning speed but is not limited to the use of the
mid-infrared (MIR) section, and the application of the beam splitter can cover
its spectral range from ultraviolet to Far-infrared segment.[2]
X-ray
crystallography characterization
The
principle of X-ray determination of protein structure is to use X-rays with a
wavelength of about 1 Å in analyzing the three-dimensional structure of
proteins. Since this wavelength is the same order of magnitude as the distance
between atoms in protein crystals, and the molecules in the crystal structure
are arranged regularly when X-rays are incident on the crystal, each atom in
the crystal emits secondary X-rays that interfere with each other and
superimpose and produce strong X-ray diffraction. For X-rays, although there is
no material that can directly gather scattered light as an objective lens to
form an image, the diffraction phenomenon of the crystal has a certain
relationship with the internal structure of the crystal; that is, the
diffraction direction is related to the shape and size of the unit cell in the
crystal. , and the intensity of diffraction is related to the arrangement and
period of heavy metal atoms in the unit cell. [3]
NMR Spectroscopy Characterization
As the main analysis method in structural biology,
nuclear magnetic resonance(NMR) spectroscopy is one of the few techniques that
can characterize the fine three-dimensional structure of proteins at the atomic
scale. With the development of high-field NMR technology, combined with protein
isotope labeling technology, it has been possible to characterize the structure
of super large proteins and protein complexes with molecular weights up to
hundreds of thousands. Compared with X-ray crystallography, NMR technology has
the advantage of being able to study the three-dimensional structure of
proteins in a state closer to the physiological environment (pH, salt
concentration, temperature, etc.). The study of protein function and the "dynamic
structure" of protein molecules is very important. NMR technology can
study the dynamic properties of proteins through the nuclear relaxation process
at the atomic level and has an incomparable effect on other technologies.
Cryo-electron
microscopy characterization
Cryo-electron microscopy is referred to as
cryo-electron microscopy or cryo-electron microscopy. This is a technology that
uses a transmission electron microscope to observe samples under low-temperature
conditions. This technology, together with X-ray crystallography
characterization and NMR technology, has laid the foundation for
high-resolution structural biology research. Compared with traditional methods
of studying the three-dimensional structure of protein molecules, such as X-ray
diffraction technology and nuclear magnetic resonance technology, cryo-electron
microscopy has the following advantages: maintaining the activity and
functional state of biological samples; The determination of the
three-dimensional structure of protein macromolecules is more suitable; the
analysis of the three-dimensional structure of protein macromolecules has the
characteristics of high throughput, fast and efficient.
X-ray-based Crystal Structural Biology
The measurement of complete
three-dimensional structure of protein and its complex assembly is the
scientific basis for studying the relationship between molecular structure and
function in life activities and revealing the physical and chemical nature of life
phenomena. The X-ray diffraction of protein and its complex crystals is one of
the main methods to study the three-dimensional fine structure of biological
macromolecules.
Penetrating with other disciplines and
especially driven by popular disciplines like structural genomics, protein
crystallography extends from analyzing the simple three-dimensional structure
of protein to studying the structure of various biological macromolecules and
complexes, and focus more on the relationship between structure and function.
In pharmaceutical R&D, protein crystallographic structure is widely and
significantly used in structure-based new drug design.
Services
High-throughput-based selection of protein crystals
Diffraction data collection in protein
synchrotron radiation
Protein-compound co-crystallization
Analysis of protein crystal structure
[1].
ChrisSutton. PROCESS. 2004(000)003:
74-75.
[2].
Jianchuan He, et al. Chinese Journal Of Clinical
Medicine. 2011(018)002: 147-149.
[3].
Qiang Xiong, et al. Carcinogenesis,
Teratogenesis & Mutagenesis. 2019(031)001: 82-85.
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