Biomedical Engineering Reference
In-Depth Information
(4) Endogenous fluorescence spectroscopy [
45
]
Endogenous fluorescence spectroscopy is commonly used to study the 3-D structure
of the protein. CD and endogenous fluorescence spectra just use certain parameters
related to the 3-D structure to assess the presence of the 3-D structure of protein.
(5) 3-D electron microscopy reconstruction [
44
]
With the continuous development of computer image-processing technology and
microscopy equipment in the last 5 years, the 3-D electron microscopy reconstruc-
tion technique became another important method for examining protein structure
following the X-ray and NMR technology.
The advantages of 3-D reconstruction techniques are as follows: (1) Molecular
morphology can be obtained directly even at a lower resolution; electron microscopy
can also give meaningful structural information. (2) It is suitable for samples
that cannot be analyzed by X-ray crystallography and NMR techniques, such as
difficultly crystallized membrane proteins and macromolecular complexes. (3) It is
suitable for capturing dynamic structural changes and is easy to combine with other
technologies to obtain high-resolution molecular complex structural information; it
contains the phase information, therefore, while determining a phase, it is direct and
more convenient than X-ray crystallography.
(6) Scanning probe microscopy (SPM)
Scanning probe microscopy is a surface analysis technique invented in the 1980s;
it is available for 3-D surface structure and dynamic processes at the nano level for
real-time, real-space observation. So, it has wide prospects in the study of structural
biology. Exoglucan CBH enzymes isolated from
Trichoderma
S38 cultures are
analyzed with SPM technology for holoenzyme molecular morphology observation.
SPM images showed that CBH I molecules are tadpole shaped, composed of a large
head and long tail, which are presumed to be core protein and substrate adsorption
domain of CBH, respectively. The length of the whole enzyme molecule is 29 nm,
and the maximum width is 9 nm [
46
].
In 1990, Rouvinen applied the X-ray diffraction method to analyze the catalytic
domain of the
Trichoderma reesei
CBH II. In 1993, Spezio crystallized and analyzed
the catalytic domain of E2 of
T. fusca
. Study of 3-D structural analysis provided
a reasonable explanation for endoglucase and exoglucase substrate specificity. In
1989, Kraulis used the NMR method to study 3-D structure of the cellulose-binding
domain (CBD) from
T. reesei
CBH I. And in 1995, Xu studied the 3-D structure of
CBD from
Cellulomonas fimi
Cex. Tormo applied the X-ray diffraction method to
study the structure of CBD from
Clostridium thermocellum
Cip in 1996 [
47
].
Spectral science reduction showed that there are five kinds of oxidation state
for LiP. Natural-state LiP contains high-spin Fe
3C
. Two electrons are oxidated by
H
2
O
2
, and LiP becomes LiP I (oxygen with iron porphyrin ring radicals containing
•
Fe
4C
). Single-electron reduction forms LiP II (oxygen with the iron porphyrin
ring containing Fe
4C
). Then, by single-electron reduction again, it returns to the
natural state. His
82
is in the cracks of the active center openings in the channel
surface, and Trp
170
is in the surface of the enzyme protein. Its electron trans-
fer may have two different pathways: substrate-His
82
-Ala
83
-Asn
84
-His
47
-heme or
C
