scholarly journals Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

2016 ◽  
Vol 72 (6) ◽  
pp. 434-442 ◽  
Author(s):  
Krzysztof P. Bzymek ◽  
Yuelong Ma ◽  
Kendra A. Avery ◽  
David A. Horne ◽  
John C. Williams
Keyword(s):  
Surface Plasmon Resonance ◽  
Disulfide Bond ◽  
Kinetic Data ◽  
Hydrophobic Pocket ◽  
Central Cavity ◽  
Cyclic Derivative ◽  
Small Perturbations ◽  
Heavy Chains ◽  
Linear Peptide ◽  
Structural Differences

Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic `pocket' and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.

Studies Toward the Total Synthesis and Stereochemical Assignment of Microspinosamide

2015 ◽  
Vol 68 (12) ◽  
pp. 1885 ◽  
Author(s):  
Gajan Santhakumar ◽  
Richard J. Payne
Keyword(s):  
Total Synthesis ◽  
Natural Product ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Chemical Ligation ◽  
Stereochemical Assignment ◽  
Linear Peptide ◽  
Cyclic Depsipeptide ◽  
Peptide Thioester ◽  
Structural Differences

Efforts toward the total synthesis and stereochemical assignment of the cyclic depsipeptide natural product microspinosamide are described. A single diastereoisomer was targeted corresponding to the predicted structure of the natural product incorporating a (2S, 3R)-β-hydroxy-p-bromophenylalanine residue. Assembly was achieved through the initial synthesis of a cyclic depsipeptide and a linear peptide thioester fragment by solid-phase peptide synthesis, followed by fusion of the two fragments through a native chemical ligation–oxidation protocol. Extensive spectroscopic analysis showed structural differences to the isolated natural product, suggesting that a diastereoisomer of microspinosamide had been synthesised. This work lays the foundation for the future synthesis of the correct diastereoisomer.

scholarly journals The light chains of kinesin-1 are autoinhibited

2016 ◽  
Vol 113 (9) ◽  
pp. 2418-2423 ◽  
Author(s):  
Yan Y. Yip ◽  
Stefano Pernigo ◽  
Anneri Sanger ◽  
Mengjia Xu ◽  
Maddy Parsons ◽  
...  
Keyword(s):  
Intramolecular Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Light Chains ◽  
Heavy Chains ◽  
Linear Peptide ◽  
Peptide Motifs ◽  
Microtubule Motor ◽  
Tetratricopeptide Repeat Domain ◽  
Cargo Binding

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLCTPR), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.

scholarly journals Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress

2021 ◽  
Author(s):  
Joseph L. Baker ◽  
Tobias Dahlberg ◽  
Esther Bullitt ◽  
Magnus Andersson
Keyword(s):  
Disulfide Bond ◽  
Pathogenic Bacteria ◽  
Alpha Helix ◽  
Covalent Bond ◽  
Dynamics Simulation ◽  
Biophysical Properties ◽  
E Coli ◽  
Steered Molecular Dynamics Simulation ◽  
Structural Differences ◽  
Class 1

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces that are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow force, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1 pili of uropathogenic E. coli (UPEC), as well as Class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Looking comprehensively at structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking β-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient shear forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili.Significance StatementAdhesion pili are often essential virulence factors for attachment of pathogenic bacteria in specific environmental niches. We provide mechanistic details of structural differences impacting the biophysical properties of pili found on bacteria in the urinary and intestinal tracts. We see that pili from urinary tract bacteria are composed of subunits optimized for their microenvironment. First, they can tolerate higher forces than intestinal pili due to a disulfide bond that limits subunit unfolding. Second, their greater flexibility is due to an α-helical motif that can unfold, absorbing force that could otherwise lead to bacteria detachment. Our work provides insight into the central role of pilus structural and biophysical properties for the sustained bacterial adherence necessary to initiate disease.

scholarly journals Exploration of insulin amyloid polymorphism using Raman spectroscopy and imaging

2019 ◽  
Author(s):  
M. Ishigaki ◽  
K. Morimoto ◽  
E. Chatani ◽  
Y. Ozaki
Keyword(s):  
Disulfide Bond ◽  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Relative Proportion ◽  
Raman Imaging ◽  
Hydroxyl Groups ◽  
Structural Differences ◽  
Α Helix ◽  
Β Sheet

AbstractWe aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature, and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90 ℃. The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to β-sheet and α-helix structures was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the β-sheet structure was higher in the case with salts, especially at a higher temperature and with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm-1) and the ratio of the tyrosine doublet R(I850⁄I826) were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis (PCA). Especially, amyloid fibrils with Na2SO4 media formed the g-g-g conformation of disulfide bond at a higher rate and without any salts; on the contrary, the g-g-g conformation was partially transformed into the g-g-t conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three-dimensional structural differences of amyloid polymorphisms. The present results indicate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.Statement of SignificanceOur results revealed three Raman markers distinguishing amyloid fibril polymorphisms caused by salt and temperature effects; the relative proportion of protein secondary structures (α–helix and β-sheet), the ratio of tyrosine doublet, and the conformational differences of disulfide bonds. The lower values of tyrosine doublet in the case with salts were interpreted as the anions rob the hydration water from proteins which induced protein misfolding. Using these parameters, Raman images captured their higher order structural differences in situ without labeling. The images of hydrogen bonds strength variations due to tyrosine doublet is believed to include significant novelty. The present results imply the potential of Raman imaging for use as a diagnostic imaging tool for tissues with amyloid-induced diseases.

scholarly journals Binding Sites of Anti-Lcr V Monoclonal Antibodies Are More Critical than the Avidities and Affinities for Passive Protection against Yersinia pestis Infection in a Bubonic Plague Model

Antibodies ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 37
Author(s):  
Kei Amemiya ◽  
Jennifer L. Dankmeyer ◽  
Sarah L. Keasey ◽  
Sylvia R. Trevino ◽  
Michael M. Wormald ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Surface Plasmon Resonance ◽  
Yersinia Pestis ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Binding Sites ◽  
Passive Protection ◽  
Linear Peptide ◽  
V Antigen ◽  
Antigen Fragment

Plague is a zoonotic disease that is caused by Yersinia pestis. Monoclonal antibodies (mAbs) that bind to the V-antigen, a virulence factor that is produced by Y. pestis, can passively protect mice from plague. An analysis of protective mAbs that bind to V-antigen was made to assess binding sites, avidities, and affinities. Anti-V mAbs were screened for their efficacy in a murine model of plague. Antigen-binding sites of protective V mAbs were determined with a linear peptide library, V-antigen fragment, competitive binding, and surface plasmon resonance. The avidities to the V-antigen was determined by ELISA, and affinities of the mAbs to the V-antigen were determined by surface plasmon resonance. The most protective mAb 7.3 bound to a unique conformational site on the V-antigen, while a less protective mAb bound to a different conformational site located on the same V-antigen fragment as mAb 7.3. The avidity of mAb 7.3 for the V-antigen was neither the strongest overall nor did it have the highest affinity for the V-antigen. The binding site of the most protective mAb was critical in its ability to protect against a lethal plague challenge.

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