OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Deng, Yibing; Zheng, Bin; Liu, Yutong; Shi, Shuting; Nie, Jingyuan; Wu, Tao; Zheng, Peng

doi:10.3791/60774

Cited by 10 publications

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“…Moreover, the demonstration here may have great potentials for further modification of gold nanoclusters. Enzymes can recognize many specific peptide sequences for ligation, such as sortase and asparaginyl ligase OaAEP1 [42,43,[55][56][57], and the use of a suitable peptide as a linker for protein immobilization may allow further characterization by single-molecule force spectroscopy [58][59][60][61][62][63][64][65][66], which may provide mechanical information about the nanocluster and immobilized protein [67][68][69][70][71]. Thus, by designing a proper peptide sequence for a first step peptide-conjugation, further functionalization and characterization of the nanoclusters can be possible [72][73][74][75][76][77].…”

Section: Discussionmentioning

confidence: 99%

“…Here, the conjugation of a lengthy peptide on Au NSs can be the first step toward site-specific protein modification. By conjugating a proper peptide with a recognition site for enzymatic connection, a target protein can be further coated on the nanoclusters via an enzymatic ligation with the peptide, such as using sortase or asparaginyl ligase (AEP) [41][42][43]. Thus, we explore here whether such small-sized Au NCs can be conjugated with a long peptide, with the ultimate goal for site-specific protein immobilization on nanocluster.…”

Section: Introductionmentioning

confidence: 99%

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Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths

Liu

Yang

et al. 2021

Nanomaterials

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The synthesis of ultra-small gold nanoclusters (Au NCs) with sizes down to 2 nm has received increasing interest due to their unique optical and electronic properties. Like many peptide-coated gold nanospheres synthesized before, modified gold nanoclusters with peptide conjugation are potentially significant in biomedical and catalytic fields. Here, we explore whether such small-sized gold nanoclusters can be conjugated with peptides also and characterize them using atomic force microscopy. Using a long and flexible elastin-like polypeptide (ELP)20 as the conjugated peptide, (ELP)20-Au NCs was successfully synthesized via a one-pot synthesis method. The unique optical and electronic properties of gold nanoclusters are still preserved, while a much larger size was obtained as expected due to the peptide conjugation. In addition, a short and rigid peptide (EAAAK)3 was conjugated to the gold nanoclusters. Their Yong’s modulus was characterized using atomic force microscopy (AFM). Moreover, the coated peptide on the nanoclusters was pulled using AFM-based single molecule-force spectroscopy (SMFS), showing expected properties as one of the first force spectroscopy experiments on peptide-coated nanoclusters. Our results pave the way for further modification of nanoclusters based on the conjugated peptides and show a new method to characterize these materials using AFM-SMFS.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths

Liu

Yang

et al. 2021

Nanomaterials

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“…For the mNT dimer experiment using Coh‐(GB1) 2 ‐mNT construct, a site‐specific protein immobilization method and a cohesion (Coh)‐dockerin (Doc) protein‐protein interaction pair as molecular handle were utilized in the AFM system to ensure that protein of interest was fully unfolded during single‐molecule experiments. [ 10a , 11 ] During the experiments, AFM tips were pressed on the substrate with a contact force of 500 pN and precisely stretched the mNT through [Coh:Doc] interaction. After recording each force‐extension curve, the tips moved horizontally by 50 nm for the subsequent measurement.…”

Section: Methodsmentioning

confidence: 99%

Single‐Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions

et al. 2022

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The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron‐sulfur protein containing a unique Fe2S2(His)1(Cys)3 metal cluster with a single Fe−N(His87) coordinating bond. This labile Fe−N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe−N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM‐SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single‐molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe−N bond.

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“…A similar strategy was used to create polymerized proteins step by step in a rationally controlled sequence. [179] WT OaAEP1 has low kinetic parameters that limit its utilization in bioconjugation of proteins, nevertheless the mutation C247A substantially increases the catalytic efficiency of the enzyme. [180] Dual Site-Specific Coupling Using a Combination of Transpeptidases: Interestingly, since each transpeptidase possesses a specific cleavage and receiving motives, it is possible to use a combination of two of them to label a protein at two distinct sites in a one-pot reaction and thus creating multimodal proteins (Figure 5C).…”

Section: Transpeptidationmentioning

confidence: 99%

Protein Engineering Strategies for Improved Pharmacokinetics

Rondon

Mahri

Morales-Yánez

et al. 2021

Adv Funct Materials

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Protein therapeutics have gained momentum in recent years and become a pillar in treating many diseases and the only choice in several ailments. Protein therapeutics are highly specific, tunable, and less toxic than conventional small drug molecules. However, reaping the full benefits of therapeutic proteins in the clinics is often hindered by issues of immunogenicity and short half-life due essentially to fast renal clearance and enzymatic degradation. Advances in polymer chemistry and protein engineering allowed overcoming some of these limitations. Strategies to prolong the half-life of proteins rely on increasing their size and stability and/or fusing them to endogenous proteins (albumin, Fc fragment of antibody) to hijack physiological pathways involved in protein recycling. On the downside, these modifications might alter therapeutic proteins structure and function. Therefore, a compromise between half-life and activity is sought. This review covers half-life extension strategies using natural and synthetic polymers as well as fusion to other proteins and sheds light on genetic engineering strategies and chemical and enzymatic reactions to achieve this goal. Promising strategies and successful applications in the clinics are highlighted. DOI: . /adfm.functions that cannot be mimicked by simple chemical compounds. Since the action of proteins is highly specific, they barely interfere with normal biological processes and cause less adverse events. Protein therapeutics are frequently derived from proteins naturally produced by the body. These agents are therefore often well tolerated and poorly immunogenic. However, proteins also suffer from significant limitations. Proteins with a molecular weight below the threshold for kidney filtration ( kDa, the size of human serum albumin) are cleared from the systemic circulation within a day. Many proteins are even cleared within a few hours or a few minutes when metabolism contributes to elimination. Therefore, therapeutic proteins need to be injected to patients several times a week (e.g., erythropoietin) or even several times a day (e.g., glucagon-like peptide-or GLP-), resulting in peaks and valleys in plasma concentrations with the alternate risks of systemic side effects and suboptimal therapeutic concentrations. Moreover, frequent administration of medication causes patient discomfort and reduces quality of life. A second limitation of proteins lies in protein immunogenicity. Foreign proteins from prokaryotes or animals might present interesting therapeutic properties in humans. However, intrinsic immunogenicity of nonhuman proteins hampers their therapeutic use in the clinic because specific antibodies generated against the foreign protein neutralize its activity and result in a loss of therapeutic efficacy over time. The unwanted immune response might even cause more serious general immune effects such as anaphylaxis.Over the last three decades, protein engineering has largely demonstrated that it can provide solutions to the limitations of natural proteins. B...

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OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Cited by 10 publications

References 0 publications

Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths

Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths

Single‐Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions

Protein Engineering Strategies for Improved Pharmacokinetics

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