bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Broad‐Based Labeling Reagents for Peptide and Protein Footprinting
Authors: Nieves Wilson , Zhang Bojie , Cheng Ming , Gross Michael 
Published: June 2021

Interactions between macromolecules in living organisms are governed by fundamental biophysical principles. Different methods to study these biophysical properties exist; including footprinting. Footprinting refers to the structural probing of interaction between macromolecules using chemical reactions ranging from HDX to reactive species such as free radicals and carbenes. Modern footprinting methods allows for a fast, specific, and precise determination of proteins' structure, interaction sites, conformational changes, folding, misfolding, and binding affinity. The complete understanding of these, in‐vitro and in the cell, is crucial for drug development and antibody synthesis for therapeutics and diagnostics. These advances contribute to the understanding of the human proteome. Methods for determining a protein footprint include Mass Spectrometry (MS), which is advantageous given its sensitivity. Covalent modification of amino acid residues coupled with MS has been useful in characterizing non‐membrane and membrane proteins; a field where current biophysical techniques are limited. Covalent modification can be achieved via Fast Photochemical Oxidation of Proteins (FPOP) using radical chemistry. Residue labeling using current radical species lacks generality or broad reactivity among amino acids. Thus, the development of novel chemical labeling methods that increases amino acid labeling are needed. To implement this, we propose the use of carbene species (double radicals), trifluoro‐methyl radicals, and chloride cations. Carbene compounds will be generated via FPOP from diazirine precursors, which were synthesized from ketone precursors. The Togni's Reagent will be used as a precursor for covalent modification through trifluoromethyl radicals and Palau's reagent will be used for modification through chloride cations. If a robust footprinting method is developed to further understand protein biophysics it would lead to further advances in the study of protein folding, protein‐protein interactions, and drug development.Support or Funding InformationAmgen Foundation. Michael Gross's Laboratory. NIH/NIGMS Biomedical Mass Spectrometry Resource.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical Analysis of Human Neuropeptide Y: Mutations in the Hairpin Core Reveal Unusual Thermal Stability Linked to Higher‐Order Self‐Association
Authors: Hopkins Mandi M. , Bain David L. 
Published: June 2021

Neuropeptide Y (NPY) is a highly conserved 36‐residue peptide, abundantly present in the central and peripheral nervous systems. NPY interacts with G‐protein coupled receptors (GPCRs) to regulate processes including energy homeostasis, affective behavior, plasticity, and memory function. The N‐terminal polyproline sequence of NPY folds back onto a C‐terminal α‐helix to form a hairpin structure. The hairpin undergoes transient unfolding to allow the monomer to interact with its target GPCRs, and to form reversible dimers in solution. Previous computational studies predicted that increasing the hydrophobicity of the hairpin core should result in a more thermodynamically stable hairpin, and decreased functional activity. To experimentally test this hypothesis, we characterized the role of two conserved tyrosines (Y20 and Y27) located within the hydrophobic core of the hairpin fold by mutating them to more hydrophobic phenylalanines. Consistent with the computational prediction, these mutations increased the thermal stability of NPY as observed by circular dichroism studies. However, mutant stability relative to wild‐type was higher than expected—melting temperatures increased by approximately 20 °C for the single mutants (Y20F and Y27F), and by 30 °C for the double mutant (Y2027F). These findings suggested that the mutations were not simply enhancing hairpin stability, but might also be driving self‐association to dimer. Using sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, we determined that the mutations indeed increased self‐association, but shifted the equilibrium toward hexamer‐like species. Notably, these later species were not unique to the NPY mutants, but were found to pre‐exist in the wild‐type population, contrary to previous reports. Collectively, the findings indicate that NPY self‐association is more complex than previously recognized, and that Y20 and Y27 may play a modulatory role in hairpin stability. Specifically, the ensemble of NPY quaternary states is adjustable by modulating the hydrophobicity of the NPY hairpin core, which may have implications for the development of new NPY‐based therapeutics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of 19F NMR to probe conformational changes of arrestin
Authors: Stout Rebecca Mae , Zhan Xuanzhi , Carroll William 
Published: June 2021

Signal specificity is achieved through specific and highly‐regulated molecular recognition. Arrestins, a small family of multi‐functional adaptor proteins, mediate surprisingly complex cellular signaling events. To elicit the appropriate cellular response, arrestins have to ensure that the correct components are linked together to prevent the formation of mismatched protein complexes. It is now clear that arrestins undergo significant conformational movements in many regulating processes; however, previous studies of arrestin conformational changes are almost completely focused on receptor‐induced conformational changes, and little is known about binding‐induced conformations that allow arrestins to accomplish their diverse signaling functions. We hypothesize that binding induced conformational changes play decisive roles in regulating the recruitment of binding partners. 19F NMR has proven to be a powerful technique in the study of protein structure and dynamics because the 19F nucleus is easily incorporated into protein through specific labeling. The chemical shift of 19F is extremely sensitive to the changes of local micro‐environment caused by the conformational movements of protein, making it possible to analyze the minor movements within a protein molecule. 19F NMR is performed by incorporating a fluorine label into the protein structure Fluorine labeling can be accomplished in a variety of ways. Here, we developed a 19F NMR‐based method to detect the conformational movements of arrestins by employing a thio‐based method to incorporate 19F to the specific residues on arrestin. We have observed the release of C‐tail in arrestin‐3 upon binding of IP‐6 (inositol hexaphosphate). The methods developed here can be employed to detect the conformational changes of arrestin with many other binding partners.Support or Funding InformationThis project is supported by NSF MRI project (1531870), TTU faculty research grant (XZ) and Student Research Grant (RS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual function of the trigger factor chaperone in nascent protein folding
Authors: Kaiser Christian , Liu Kaixian , Maciuba Kevin 
Published: June 2021

Multi‐domain proteins – constituting a large group in all proteomes – often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. We have used optical tweezers to study the folding of elongation factor G (EF‐G), a model multi‐domain protein, as it emerges from the ribosome. We find that the N‐terminal G‐domain in nascent EF‐G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non‐native state. This non‐native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and non‐productive interactions among unfolded domains are efficiently prevented by the ribosome‐binding chaperone trigger factor. Thus, our single‐molecule measurements of multi‐domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding early folding defects is crucial, since they can propagate and result in misfolding of the entire protein. Our experiments define the folding pathway for a complex multi‐domain protein and shed light on the molecular mechanism employed by molecular chaperones to ensure productive and efficient folding.Support or Funding InformationCMK acknowledges support by the Pew Scholars Program in the Biomedical Sciences and the National Institute of General Medical Sciences (1R01GM121567)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effectiveness of in silico Engineering of the β‐glucosidase B Enzyme
Authors: Boulanger Kyle R. , Hall Bonnie L. 
Published: June 2021

Enzymes are used in a wide range of commercial and medical applications, and it is desirable for these enzymes to have maximum efficiency. A common approach to engineering a more efficient enzyme is generating random mutations and selecting those that increase enzyme activity in vitro. In silico design utilizes molecular modeling software to more rapidly predict structure‐function relationships, thus identifying more efficient enzymes without massive in vitro screening efforts. The drawback with these predictions is a lack of accuracy, yielding correct results as low as ten percent of the time (Carlin, 2016). To improve the algorithms used for molecular modeling, a large pool of enzyme mutants would need to be analyzed both in silico and simultaneously characterized kinetically under consistent experimental conditions. We are characterizing the BglB protein, as it is easily overexpressed and its efficiency can be measured using a simple colorimetric assay. We have engineered a pool of BglB mutants in silico using FoldIt, and compared the computational predictions with the in vitro kinetic results. Based on kcat and Km values for the various BglB mutants, we found FoldIt was able to correctly predict structure‐function relationships for BglB approximately 40% of the time. The remaining 60% of the time, the software predictions did not match the in vitro results. These BglB data, as a part of the larger SEEK database, can help refine the algorithms used in the FoldIt modeling software. Hopefully, molecular modeling will eventually be able to accurately predict structure‐function results in silico, saving both time and money in the enzyme engineering process.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineer Alkane Hydroxylating Properties into Phenol Hydroxylase: A Look at the 2nd Coordination Sphere
Authors: Guillen Erik , Sazinsky Matthew 
Published: June 2021

Bacterial Multicomponent Monooxygenases (BMMs) are diiron enzymes characterized by the ability to hydroxylate a variety of hydrocarbon substrates. BMMs are naturally found in bacteria whose natural source of energy is derived from small organic compounds requiring initial hydroxylation for bacterial aerobic respiration. BMMs are of interest because the substrate transformations they perform can be useful in removing atmospheric greenhouse gases (i.e. methane) from the environment and such biocatalysts have potential applications in industrial organic syntheses. Phenol hydroxylase (PH) from Pseudomonas sp OX1 is a BMM family member essential for carrying out the oxidation of phenol to catechol but unlike the highly homologous soluble methane monoxygenase, it is unable to carry out oxidation on alkanes. Various studies suggest that the diiron coordination sphere in PH and sMMO are nearly identical, however, it is not understood how the protein scaffolds tune their reactivity toward different substrates. Here we present our efforts to explore the effects of the 2nd coordination sphere on reactivity and report on the development a screen to that would allow us search for alkane‐hydroxylating properties among a library of PH mutants.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mini‐Ins: a Monomeric Human Insulin Inspired From Cone Snail Venom Peptides
Authors: Chou Danny 
Published: June 2021

For people with diabetes, maintaining blood glucose levels within the nondiabetic range is effective in delaying or even preventing long‐term complications, but achieving near normoglycemia is still challenging for these patients. This is due to the fact that the current fast‐acting insulin, Humalog, still has relatively slow absorption after subcutaneous injection which leads to delayed insulin response and sub‐optimal control of glycemia. An even faster‐acting insulin requires a monomeric insulin molecule. However, the region of the insulin molecule responsible for self‐association is also responsible for insulin receptor binding. Therefore, despite millions of dollars invested, a truly monomeric, ultrafast‐acting insulin remains to be invented. In this project, we take a bio‐inspired approach to utilize a venom insulin as a potential candidate with ultrafast‐acting properties. Recently, we and collaborators reported that a cone snail venom insulin, Con‐Ins G1, is monomeric in nature due to its shortened B‐chain. Furthermore, Con‐Ins G1 is bioactive toward human insulin signaling, albeit with 20‐fold reduction in potency. Learning from the structural insights, we performed a series of structure‐activity relationship studies to identify the key mutations from Con‐Ins G1 that lead to increased bioactivity. We have identified A8H, A9R, B10E, B20Y Des‐octapeptide (B23–30) insulin “mini‐Ins” as the shortest bioactive human insulin analog discovered to date. In vitro and in vivo experiments demonstrated equal biological potencies between native insulin, Humalog and mini‐Ins. The discovery of mini‐Ins demonstrates a promising therapeutic potential as a monomeric insulin.Support or Funding InformationThis work was supported by Utah Science Technology and Research Agency and the Margolis Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of Polymer Based Protein Engineering and ATRP to Modulate Substrate Size Specificity of Avidin
Authors: Winegardner Nicole P. , Adams Haili , Lucas Amber , Kaupbayeva Bibifatima , Murata Hironobu , Russell Alan S. , Minden Jonathan S. 
Published: June 2021

Avidin and biotin interact very strongly and are commonly used to tag and purify proteins or peptides for proteomic analysis. Using a pH dependent reversible biotin tag that targets primary amines, all the proteins can be tagged at a slightly basic pH. After labeling proteins with the biotin tag, any excess unreacted biotin left over in solution must be dialyzed away to prevent competition for binding spots on the Avidin beads. Tagged proteins can then be bound to a solid surface Avidin bead and contaminants washed away. Proteins can then be released from the beads in their original unmodified state by lowering the pH. While this workflow has been shown to be very useful for purifying proteomes, the requirement for dialysis after the initial tagging step makes this workflow laborious and difficult to automate. This step is time consuming, can result in protein loss or precipitation, and makes automation impossible. In this study we seek to utilize polymer based protein engineering to circumvent this issue.Atom transfer radical polymerization (ATRP) has been utilized in recent years to specifically modify and fine tune the behavior of proteins. By adding polymers of a specific composition and length in targeted areas of a protein, the behavior of a protein can be modified. In this study, we utilized ATRP to grow a dense polymer network off the surface of Avidin to act as a molecular sieve; The large polymers allow small molecules of biotin to bind to the binding site, while larger biotinylated proteins are excluded. The resulting “Caged Avidin” can then be used in the proteome purification workflow in the place of dialysis by binding and sequestering any unreacted free biotin, allowing the entire labeling, binding, washing, and eluting workflow to take place in a single tube with no bias and no sample loss. This can revolutionize sample preparation in biomarker and drug discovery by allowing for automation, and thus improved yield and reliability, of sample preparation.Support or Funding InformationThis work was supported in part by the Center for Polymer Based Protein Engineering at Carnegie Mellon UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fast and Accurate Evaluation of Oxidation‐Induced Destabilization of mAbs
Authors: Piatti Patricia , Mohamadi Mariam , Tschammer Nuška , Breitsprecher Dennis , Fung Peter A. 
Published: June 2021

The applicability of monoclonal antibodies (mAbs) in therapeutic research continues to rise — they now account for almost 50% of protein‐based drugs. Monitoring their quality as well as binding properties are critical as these parameters provide insight into mAb functionality and efficacy as potential drugs.Here we study trastuzumab, a monoclonal antibody that has been used to successfully treat patients with certain forms of breast cancer. Trastuzumab acts by binding to and interfering with the HER2/neu receptor in cancer patients. Using two complementary technologies, we examine how targeted oxidation affects trastuzumab structure and therefore its binding capabilities to protein A. First, a rapid analysis of oxidation‐induced changes in mAb folding and stability was performed. The same samples were then analyzed to determine how oxidation compromises mAb interactions. Oxidational stress directly correlated with weaker binding affinities of the Fc region towards protein A, showcasing fast and accurate characterization of trastuzumab sample quality.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cooperative Treatment of Breast Cancer Using an Irinotecan/IR‐820 Co‐loaded Hollow Mesoporous Silica Nanoparticles Nanoplatform
Authors: Li Tingting , Geng Yue , Chen Zhongyuan , Yang Hong , Wu Chunhui , Liu Yiyao 
Published: June 2021

BackgroundNanomaterial‐based multimodal systems are promising candidates for exerting synergistic therapeutic effects in cancer therapy.1 Combination therapy through different mechanisms can cooperatively inhibit cancer progression and overcome the limitations of monotherapies.2 In this study, we used hollow mesoporous silica nanoparticles (HMSNs) as a biocompatible carrier to co‐load chemotherapy drugs Irinotecan and near‐infrared (NIR) IR‐820 dye, which enhanced anti‐tumor efficacy by combining chemotherapy and phototherapy.MethodsHollow mesoporous silica nanoparticles (HMSNs) were prepared by an established method 1, then IR 820 and Irinotecan were loaded into the HMSNs by electrostatic attracting method. The successful synthesis of HMSNs/Irinotecan/IR820 (HMII) nanocomplex were confirmed by Fourier Transform Infrared (FTIR) spectroscopy and Fluorescence spectra. Confocal laser scanning microscopy (CLSM) was used to detect the cellular uptake of HMII. The photothermal conversion efficiency and anti‐tumor efficiency in mouse mammary cells (EMT‐6) bearing mice were further evaluated.ResultsThe mean diameter of HMII were around 80 nm, which possessed the advantages of homogenous particle size, high stability, and good safety. The results demonstrated that HMII enhanced the delivery of Irinotecan and IR‐820 into EMT‐6 cells. HMII generated a high temperature upon a NIR laser irradiation (808 nm), and showed higher therapeutic efficacy in EMT‐6‐bearing mice compared to either HMII without laser or free drug with a laserConclusionBy combining chemotherapy and phototherapy (PTT), HMII efficiently inhibited the growth of breast cancer in mice. This nanosystem provides promising applicability for combination therapy of breast cancer.Support or Funding InformationWe would like to thank the National Natural Science Foundation of China (81671821, 81471785, 31470959, 31470906, and 11502049)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.