China
Africa
Explore chapters and articles related to this topic
Translation
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Finally, Hobson and Uhlenbeck (2006) performed alanine scanning of the MS2 coat and revealed protein-phosphate contacts involved in thermodynamic hot spots. Since the co-crystal structure of the MS2 coat dimer with the RNA operator found eight amino acid side chains contacting seven of the RNA phosphates, these eight amino acids and five nearby control positions were individually changed to an alanine residue, and the binding affinities of the mutant proteins to the RNA were determined. In general, the data agreed well with the crystal structure and previous RNA modification data. Interestingly, the amino acid residues that were energetically most important for complex formation clustered in the middle of the RNA binding interface, forming thermodynamic hot spots, and were surrounded by energetically less relevant amino acids. In order to evaluate whether or not a given alanine mutation caused a global change in the RNA-protein interface, the affinities of the mutant proteins to RNAs containing one of 14 backbone modifications spanning the entire interface were determined. In three of the six protein mutations tested, the thermodynamic coupling between the site of the mutation and the RNA groups that could be even more than 16 Å away was detected.
Melanoma Growth Stimulatory Activity: Physiology, Biology, Structure/Function, and Role in Disease
Published in Richard Horuk, Chemoattractant Ligands and Their Receptors, 2020
Ann Richmond, Rebecca L. Shattuck
Alanine scanning mutagenesis studies have identified several additional amino acid residues within the IL-8 receptor A which are essential for IL-8 binding.95 These studies demonstrated that Glu-275 and Arg-280 located within the third extracellular loop are required for retention of ligand binding. Equivalent amino acid residues are also present in the third extracellular loop of the B receptor and presumably are involved in MGSA/GRO binding as well as in IL-8 binding to the B receptor. In addition, Asp-11 is critical, though mutation to Glu or Lys does not affect the affinity of the ligand receptor interaction.95
Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes
Published in mAbs, 2021
Pavel V. Bondarenko, Andrew C. Nichols, Gang Xiao, Rachel Liuqing Shi, Pik Kay Chan, Thomas M Dillon, Fernando Garces, David J. Semin, Margaret S. Ricci
The paratope mapping technique described here is somewhat similar to alanine-scanning mutagenesis,20–22 but with the following key differences. Instead of mutations, side chains of amino acid residues are altered by chemical modifications, which is a simpler process. Also, instead of one-by-one binding to target measurements, the described method allows parallel, large-scale paratope and potentially epitope mapping. Although alanine-scanning mutagenesis can potentially provide epitope/paratope information at the amino acid level, it is a rather lengthy and laborious process, and it suffers from the uncertainty of not knowing whether the mutation genuinely affected a key interacting residue or just disrupted the folding of the mutated protein.15 And yet, scanning mutagenesis remains the most reliable and preferred method for paratope and epitope mapping. Our technique encounters similar uncertainty because the side chains are modified before binding. As our studies of several antibody-target molecules indicate, the method provides useful information, and long-range, allosteric modifications were not common during paratope mapping. This is probably because antibody binding sites (paratope) are typically located in CDRs, which are unfolded, unstructured, flexible loops. Modifications also preferentially take place on unfolded, unstructured, flexible loops (which are mainly CDRs). Therefore, modifications should not affect folding, and cause long-range impacts, but rather differentiate critical and non-critical modifications in CDRs.
High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies
Published in mAbs, 2019
T. Noelle Lombana, Marissa L. Matsumoto, Jack Bevers III, Amy M. Berkley, Evangeline Toy, Ryan Cook, Yutian Gan, Changchun Du, Peter Liu, Paul Schnier, Wendy Sandoval, Zhengmao Ye, Jill M. Schartner, Jeong Kim, Christoph Spiess
Thus, limited by the intrinsic trade-off between the resolution and the throughput of existing epitope mapping techniques (Fig. S1), we developed the glycosylation-engineered epitope mapping (GEM) method to provide both high-throughput and high-resolution epitope mapping. GEM uses mutagenesis of single residues at strategic locations within an antigen sequence to introduce an N-linked glycosylation site on the solvent-exposed surface of the protein. When GEM mutants are recombinantly produced in mammalian cells, N-linked glycans are added to the mutation site and provide steric hindrance to antibodies that bind epitopes containing this or neighboring residues. GEM combines the high-throughput nature of competition binding with the high-resolution sequence information of alanine scanning, but it requires far fewer proteins to be produced compared to alanine scanning. As a proof of concept, we applied GEM to the well-characterized anti-human epidermal growth factor receptor-2 (HER2) antibodies, hum4D5 and hum2C4, and their known epitopes on HER2. We demonstrate that GEM epitopes are consistent with mutational analysis and epitopes defined by X-ray crystal structures. We then applied the GEM method to characterize a panel of anti-MICA/B antibodies. GEM allowed rapid and detailed mapping of an epitope that overlaps with known MICA cleavage sites and correlates with strong shedding inhibition of MICA/B from tumor cells. Furthermore, targeting this specific epitope results in surface stabilization of MICA/B and prevention of tumor growth in vivo.
Disrupting the intramolecular interaction between proto-oncogene c-Src SH3 domain and its self-binding peptide PPII with rationally designed peptide ligands
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Peng Zhou, Shasha Hou, Zhengya Bai, Zhongyan Li, Heyi Wang, Zheng Chen, Yang Meng
It is shown in Figure 4(a) that the side-chains of charged/polar residues Lys249 and Thr252 point directly to the peptide-binding pocket of SH3 domain; nonbonded analysis also found that the two residues can separately form a salt bridge and a hydrogen bond with the domain residues Asp91 and Arg95, respectively (Figure 4(b)). As can be seen in Figure 4(a), both the two residues locate in an amphipathic microenvironment that are surrounded by a number of nonpolar/aromatic domain residues (Tyr90, Tyr92, Pro133, Tyr136 and Trp118) and polar/charged domain residues (Asp91, Arg95 and Asn135). In this respect, the peptide Lys249 and Thr252 are suggested as anchor residues that should interact effectively with the domain. This can be further solidified by computational alanine scanning. As shown in Figure 4(c), most of native PPII residues (Ser248, Lys249, Pro250, Thr252, Gln253 and Leu255) play a favourable role (ΔΔGAla>0) in the domain–peptide binding, where the Lys249 and Thr252 are most important as mutation of them to neutral Ala residue would cause >1 kcal/mol binding free energy loss for the peptide ligand. In addition, there are also few residues (Thr247, Gln251 and Gly254) that contribute unfavourably (ΔΔGAla<0) to the peptide binding, albeit the unfavourable effect is very modest.