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Nanoparticles Carrying Biological Molecules
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Suryani Saallah, Wuled Lenggoro
Generally, physical conjugation offers a simple and rapid route to bind biomolecule to the NP and requires minimal modification steps (Saallah and Lenggoro, 2018). Thus, the biomolecules’ functionalities can be preserved. Nevertheless, this method suffers from weak binding, random orientation, high likelihood of desorption, and poor reproducibility (Liebana and Drago, 2016). Such drawbacks can be overcome by introducing specific functional groups or targeting ligands to the nanoparticles through affinity interactions. The most well-known example in the last several decades is the avidin–biotin system. Avidin comprises four identical subunits that provide four binding pockets which specifically recognize and bind to biotin, resulting in a strong and stable interaction with a dissociation constant, KD, of the order of 10–15 M. The combination of basic pI and carbohydrate content, however, results in nonspecific binding as observed in several applications (Sapsford et al., 2013). Alternatively, streptavidin, a non-glycosylated homologous tetrameric protein displaying similar affinity to biotin, can be used as avidin analog (Saallah and Lenggoro, 2018). The mechanism of the most widely applied non-covalent interactions is illustrated in Figure 4.7.
Playing “Nano-Lego”: VNPs as Building Blocks for the Construction of Multi-Dimentional Arrays
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Multi-layered thin films of CCMV and CPMV have been constructed using the biotin-streptavidin system (Steinmetz et al., 2008a, 2006; Suci et al., 2006). To achieve this, biotinylated VNPs are immobilized on a solid support; biotinylated VNPs can be bound on a streptavidin-coated substrate or Cys-added biotinylated mutants bound on a gold surface. Next, strepatividin is added. Streptavidin is a tetrameric protein and hence has four binding sites for biotin. The multivalency of the VNPs and streptavidin allows interconnection of the VNP layers. A second biotinylated VNP can then be deposited, followed by a further streptavidin layer, and so on. This process can be repeated until the desired number of VNP layers is deposited.
FloDots for Bioimaging and Bioanalysis
Published in Wolfgang Sigmund, Hassan El-Shall, Dinesh O. Shah, Brij M. Moudgil, Particulate Systems in Nano- and Biotechnologies, 2008
G. Yao, Y. Wu, D.L. Schiavone, L. Wang, W. Tan
Physical absorption is primarily used to modify FloDots with avidin molecule (12,14). Avidin, synthesized in the hen oviduct, is a glycoprotein of MW 68,000 daltons with an isoelectric point (pI) of 10.5 (38). It is a tetrameric protein composed of four identical subunits, each of which has one binding site for biotin. Biotin-avidin interaction has been widely applied for bioconjugation because (a) this interaction is the strongest known noncovalent biological coupling with a constant of 1015 L/mol (39), and (b) biotin is a small molecule and can be easily conjugated with other biomolecules without significantly changing their biological properties.
Purification and biochemical characterization of pullulanase produced from Bacillus sp. modified by ethyl-methyl sulfonate for improved applications
Published in Preparative Biochemistry & Biotechnology, 2023
Oladipo O. Olaniyi, Blessing Oriade, Olusola T. Lawal, Adeyemi O. Ayodeji, Yetunde O. Olorunfemi, Festus O. Igbe
The molecular are found to be within the range of 35–70 kDa molecular masses so far reported and this may also reveal monomeric properties of the proteins[43]. The slight difference in molecular weight obtained by the wild and mutant Bacillus sp β- mannanase might be due to modifications of genes that have taken place at the loci during evolution and then led to different protein structure, though they are recognized as similar proteins[44]. Olaniyi et al.[4] reported Bacillus safensis pullulanase molecular weight to be 42 kDa. There have been reports of 54 kDa to 143 kDa[37] while Khabade et al., [29] also observed molecular weight of about 100 kDa from thermophilic bacteria. Similarly, extracellular pullulanase with a molecular mass of 120 kDa was purified from Thermoanaerobium Tok6-B1[37]. Ramdas et al.[44] reported that most pullulanases isolated so far have a molecular weight of 98 KDa. However, some pullulanases were documented to be built up of large subunits known as tetrameric protein (100 kDa to 125 kDa)[43]. The variation in molecular weights could be attributed to isozymic properties as well as types of amino acid residues present in the enzymes[13].
Enhancement of protein flocculant properties through carboxyl group methylation and the relationship with protein structural changes
Published in Journal of Dispersion Science and Technology, 2021
Rafael A. Garcia, Phoebe X. Qi, Matthew Essandoh, Lorelie P. Bumanlag
Native BHb consists of four globin chains, two α and two β chains. Each symmetrical (αβ) dimer contains three Trp residues, totaling six Trp residues in the tetrameric protein. It has been reported that the fluorescence of native BHb is often difficult to detect due to the severe quenching of Trp fluorescence by the neighboring heme groups.[43] Our fluorescence experiments showed low fluorescence intensity for the native BHb (Figure 3) at λem = 340 nm. BHb from reaction time 0 showed an eight-fold increase in the Trp intrinsic fluorescence intensity, consistent with the finding of heme pocket disruption as detected by UV-Vis absorption spectroscopy. Although the magnitude of the fluorescence continued to rise slowly as the reaction proceeded, there was little change in the position of the peak, demonstrating that the tertiary contacts associated with the six Trp residues were stabilized throughout the course of the reaction. This is also consistent with the secondary structural analysis (as above).
Electron paramagnetic resonance of globin proteins – a successful match between spectroscopic development and protein research
Published in Molecular Physics, 2018
Sabine Van Doorslaer, Bert Cuypers
The CW-EPR spectra of NO-ligated human haemoglobin (HbNO) are even more complex than those of MbNO, since the α- and β-chains of the protein exhibit a different NO-binding behaviour [124,130,131]. Furthermore, the relaxed (R) and tense (T) quaternary structure of the tetrameric protein influences the NO binding. In the NO-ligated T state, the α-haems are 5-coordinate, while the β-haems are 6-coordinate [130]. In the R state, the α(β)-chains exhibit mainly an R6(A6) form at low temperatures. This was exploited in an X-band three-pulse ESEEM and HYSCORE study [132]. This investigation allowed determining the hyperfine and nuclear quadrupole parameters of the haem 14N nuclei. The hyperfine couplings of these nitrogen nuclei were somewhat higher for the A6 form than for the R6 form, but both were below 4 MHz. An additionally observed hyperfine interaction of ∼5 MHz was tentatively assigned to the Nϵ nitrogen of the distal HisE7. Similar values followed from QM/MM computations on MbNO for this nitrogen [122], suggesting that the earlier assignment could be correct.