scholarly journals Monitoring the binding and insertion of a single transmembrane protein by an insertase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pawel R. Laskowski ◽  
Kristyna Pluhackova ◽  
Maximilian Haase ◽  
Brian M. Lang ◽  
Gisela Nagler ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Transmembrane Protein ◽  
Single Molecule Force Spectroscopy ◽  
Conformational Variability ◽  
General Relevance ◽  
Helical Hairpin ◽  
Domains Of Life ◽  
Helical Proteins ◽  
Dynamics Simulations

AbstractCells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.

scholarly journals Insertion and folding pathways of single membrane proteins guided by translocases and insertases

2019 ◽  
Vol 5 (1) ◽  
pp. eaau6824 ◽  
Author(s):  
Tetiana Serdiuk ◽  
Anja Steudle ◽  
Stefania A. Mari ◽  
Selen Manioglu ◽  
H. Ronald Kaback ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Random Order ◽  
Lactose Permease ◽  
Single Molecule Force Spectroscopy ◽  
Essential Components ◽  
Protein Biogenesis ◽  
Folding Pathways ◽  
Helical Proteins ◽  
Insertion Process

Biogenesis in prokaryotes and eukaryotes requires the insertion of α-helical proteins into cellular membranes for which they use universally conserved cellular machineries. In bacterial inner membranes, insertion is facilitated by YidC insertase and SecYEG translocon working individually or cooperatively. How insertase and translocon fold a polypeptide into the native protein in the membrane is largely unknown. We apply single-molecule force spectroscopy assays to investigate the insertion and folding process of single lactose permease (LacY) precursors assisted by YidC and SecYEG. Both YidC and SecYEG initiate folding of the completely unfolded polypeptide by inserting a single structural segment. YidC then inserts the remaining segments in random order, whereas SecYEG inserts them sequentially. Each type of insertion process proceeds until LacY folding is complete. When YidC and SecYEG cooperate, the folding pathway of the membrane protein is dominated by the translocase. We propose that both of the fundamentally different pathways along which YidC and SecYEG insert and fold a polypeptide are essential components of membrane protein biogenesis.

scholarly journals Mechanisms of Nanonewton Mechanostability in a Protein Complex Revealed by Molecular Dynamics Simulations and Single-Molecule Force Spectroscopy

2019 ◽  
Vol 141 (37) ◽  
pp. 14752-14763 ◽  
Author(s):  
Rafael C. Bernardi ◽  
Ellis Durner ◽  
Constantin Schoeler ◽  
Klara H. Malinowska ◽  
Bruna G. Carvalho ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Single Molecule ◽  
Protein Complex ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Dynamics Simulations

scholarly journals Molecular mechanism of extreme mechanostability in a pathogen adhesin

Science ◽  
2018 ◽  
Vol 359 (6383) ◽  
pp. 1527-1533 ◽  
Author(s):  
Lukas F. Milles ◽  
Klaus Schulten ◽  
Hermann E. Gaub ◽  
Rafael C. Bernardi
Keyword(s):  
Single Molecule ◽  
Mechanical Stability ◽  
Force Spectroscopy ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Peptide Backbone ◽  
Human Fibrinogen ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Dynamics Simulations

High resilience to mechanical stress is key when pathogens adhere to their target and initiate infection. Using atomic force microscopy–based single-molecule force spectroscopy, we explored the mechanical stability of the prototypical staphylococcal adhesin SdrG, which targets a short peptide from human fibrinogen β. Steered molecular dynamics simulations revealed, and single-molecule force spectroscopy experiments confirmed, the mechanism by which this complex withstands forces of over 2 nanonewtons, a regime previously associated with the strength of a covalent bond. The target peptide, confined in a screwlike manner in the binding pocket of SdrG, distributes forces mainly toward the peptide backbone through an intricate hydrogen bond network. Thus, these adhesins can attach to their target with exceptionally resilient mechanostability, virtually independent of peptide side chains.

Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding

Langmuir ◽  
2017 ◽  
Vol 33 (41) ◽  
pp. 10851-10860 ◽  
Author(s):  
Kartik Srinivasan ◽  
Suvrajit Banerjee ◽  
Siddharth Parimal ◽  
Lars Sejergaard ◽  
Ronen Berkovich ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Single Molecule ◽  
Specific Binding ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Dynamics Simulations

scholarly journals Modeling the structure of the frameshift stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers

2020 ◽  
Author(s):  
Sara Ibrahim Omar ◽  
Meng Zhao ◽  
Rohith Vedhthaanth Sekar ◽  
Sahar Arbabi Moghadam ◽  
Jack A. Tuszynski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Models ◽  
Viral Proteins ◽  
Single Molecule Force Spectroscopy ◽  
Ribosomal Frameshifting ◽  
Structural Prediction ◽  
Prediction Tools ◽  
Dynamics Simulations ◽  
Rna Pseudoknot ◽  
Stem Structure

The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses −1 programmed ribosomal frameshifting (−1 PRF) to control the relative expression of viral proteins. As modulating −1 PRF can inhibit viral replication, the RNA pseudoknot stimulating −1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by μs-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through the junction formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting −1 PRF in SARS-CoV-2.

Mechanical unfolding of a β-barrel membrane protein by single-molecule force spectroscopy

2020 ◽  
Author(s):  
Hui Chen ◽  
Guangtao Song ◽  
Yong Zhang ◽  
Dongchun Ni ◽  
Xinwei Zhang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy

scholarly journals Computational and single-molecule force studies of a macro domain protein reveal a key molecular determinant for mechanical stability

2010 ◽  
Vol 107 (5) ◽  
pp. 1989-1994 ◽  
Author(s):  
Dora L. Guzmán ◽  
Arlo Randall ◽  
Pierre Baldi ◽  
Zhibin Guan
Keyword(s):  
Molecular Dynamics ◽  
Single Molecule ◽  
Mechanical Stability ◽  
Single Molecule Force Spectroscopy ◽  
Load Bearing ◽  
Molecular Determinant ◽  
Macro Domain ◽  
Domain Protein ◽  
Dynamics Simulations ◽  
High Mechanical Stability

Resolving molecular determinants of mechanical stability of proteins is crucial in the rational design of advanced biomaterials for use in biomedical and nanotechnological applications. Here we present an interdisciplinary study combining bioinformatics screening, steered molecular dynamics simulations, protein engineering, and single-molecule force spectroscopy that explores the mechanical properties of a macro domain protein with mixed α + β topology. The unique architecture is defined by a single seven-stranded β-sheet in the core of the protein flanked by five α-helices. Unlike mechanically stable proteins studied thus far, the macro domain provides the distinct advantage of having the key load-bearing hydrogen bonds (H bonds) buried in the hydrophobic core protected from water attacks. This feature allows direct measurement of the force required to break apart the load-bearing H bonds under locally hydrophobic conditions. Steered molecular dynamics simulations predicted extremely high mechanical stability of the macro domain by using constant velocity and constant force methods. Single-molecule force spectroscopy experiments confirm the exceptional mechanical strength of the macro domain, measuring a rupture force as high as 570 pN. Furthermore, through selective deletion of shielding peptide segments, we examined the same key H bonds under hydrophilic environments in which the β-strands are exposed to solvent and verify that the high mechanical stability of the macro domain results from excellent shielding of the load-bearing H bonds from competing water. Our study reveals that shielding water accessibility to the load-bearing strands is a critical molecular determinant for enhancing the mechanical stability of proteins.

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