Amino acid binding to nanotube: Simulation of membrane protein channels by computational methods

Until recently, a modest number of approx. 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. In the present study, we demonstrate the lysosomal localization of the mouse orthologue of the human C1orf85 protein, which has been termed kidney-predominant protein NCU-G1 (GenBank® accession number: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue C-terminal tail. Its lysosomal localization was confirmed using immunofluorescence with a C-terminally His-tagged NCU-G1 and the lysosomal marker LAMP-1 (lysosome-associated membrane protein-1) as a reference, and by subcellular fractionation of mouse liver after a tyloxapol-induced density shift of the lysosomal fraction using an anti-NCU-G1 antiserum. In transiently transfected HT1080 and HeLa cells, the His-tagged NCU-G1 was detected in two molecular forms with apparent protein sizes of 70 and 80 kDa, and in mouse liver the endogenous wild-type NCU-G1 was detected as a 75 kDa protein. The remarkable difference between the apparent and the calculated molecular masses of NCU-G1 was shown, by digesting the protein with N-glycosidase F, to be due to an extensive glycosylation. The lysosomal localization was impaired by mutational replacement of an alanine residue for the tyrosine residue within the putative sorting motif.

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Cysteine-based fluorescence “turn-on” sensors for Cu2+ and Ag+

RSC Advances ◽

10.1039/c4ra10936h ◽

2014 ◽

Vol 4 (99) ◽

pp. 56539-56542 ◽

Cited By ~ 3

Author(s):

V. Haridas ◽

P. P. Praveen Kumar ◽

Cherumuttathu H. Suresh

Keyword(s):

Amino Acid ◽

Computational Methods ◽

Metal Ion ◽

Ion Binding ◽

Metal Ion Binding ◽

Turn On ◽

Fluorescence Turn On

Amino acid cysteine was transformed to fluorescent turn-on sensors for Cu+2 and Ag+. The metal ion binding was studied in detail by spectroscopic, microscopic, calorimetric and computational methods.

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An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54473-2 ◽

1991 ◽

Vol 266 (34) ◽

pp. 23135-23141 ◽

Cited By ~ 1

Author(s):

W. Schlumbohm ◽

T. Stein ◽

C. Ullrich ◽

J. Vater ◽

M. Krause ◽

...

Keyword(s):

Amino Acid ◽

Reaction Center ◽

Gramicidin S ◽

Amino Acid Binding

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Perspectives on Membrane Protein Insertion, Protein–Bilayer Interactions, and Amino Acid Side Hydrophobicity

The Journal of General Physiology ◽

10.1085/jgp.200709792 ◽

2007 ◽

Vol 129 (5) ◽

pp. 351-352 ◽

Cited By ~ 9

Author(s):

Olaf Sparre Andersen

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Protein Insertion ◽

Membrane Protein Insertion

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The development and amino acid binding ability of nano-materials based on azo derivatives: Theory and experiment

Materials Science and Engineering C ◽

10.1016/j.msec.2014.01.049 ◽

2014 ◽

Vol 38 ◽

pp. 101-106 ◽

Cited By ~ 7

Author(s):

Xuefang Shang ◽

Jinge Du ◽

Wancai Yang ◽

Yun Liu ◽

Zhiyuan Fu ◽

...

Keyword(s):

Amino Acid ◽

Nano Materials ◽

Binding Ability ◽

Azo Derivatives ◽

Amino Acid Binding ◽

Theory And Experiment

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Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m703369200 ◽

2007 ◽

Vol 282 (46) ◽

pp. 33831-33844 ◽

Cited By ~ 18

Author(s):

Yoshinori Kashiwayama ◽

Kota Asahina ◽

Masashi Morita ◽

Tsuneo Imanaka

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Protein Targeting ◽

Fluorescent Protein ◽

Chinese Hamster Ovary Cells ◽

Intracellular Localization ◽

Chinese Hamster ◽

Transmembrane Domains ◽

Peroxisomal Membrane ◽

Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

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Protein structure prediction and design in a biologically-realistic implicit membrane

10.1101/630715 ◽

2019 ◽

Author(s):

Rebecca F. Alford ◽

Patrick J. Fleming ◽

Karen G. Fleming ◽

Jeffrey J. Gray

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Structure Prediction ◽

Protein Design ◽

Structure Prediction ◽

De Novo ◽

Computational Design ◽

Amino Acid Distribution

ABSTRACTProtein design is a powerful tool for elucidating mechanisms of function and engineering new therapeutics and nanotechnologies. While soluble protein design has advanced, membrane protein design remains challenging due to difficulties in modeling the lipid bilayer. In this work, we developed an implicit approach that captures the anisotropic structure, shape of water-filled pores, and nanoscale dimensions of membranes with different lipid compositions. The model improves performance in computational bench-marks against experimental targets including prediction of protein orientations in the bilayer, ΔΔG calculations, native structure dis-crimination, and native sequence recovery. When applied to de novo protein design, this approach designs sequences with an amino acid distribution near the native amino acid distribution in membrane proteins, overcoming a critical flaw in previous membrane models that were prone to generating leucine-rich designs. Further, the proteins designed in the new membrane model exhibit native-like features including interfacial aromatic side chains, hydrophobic lengths compatible with bilayer thickness, and polar pores. Our method advances high-resolution membrane protein structure prediction and design toward tackling key biological questions and engineering challenges.Significance StatementMembrane proteins participate in many life processes including transport, signaling, and catalysis. They constitute over 30% of all proteins and are targets for over 60% of pharmaceuticals. Computational design tools for membrane proteins will transform the interrogation of basic science questions such as membrane protein thermodynamics and the pipeline for engineering new therapeutics and nanotechnologies. Existing tools are either too expensive to compute or rely on manual design strategies. In this work, we developed a fast and accurate method for membrane protein design. The tool is available to the public and will accelerate the experimental design pipeline for membrane proteins.

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Resolving the Dynamic Non-Covalent Interaction inside Membrane Protein Channel by Single-Molecule Interaction Spectrum

10.26434/chemrxiv.7251683 ◽

2018 ◽

Author(s):

Meng-Yin Li ◽

Yi-Lun Ying ◽

Xi-Xin Fu ◽

Jie Yu ◽

Shao-Chuang Liu ◽

...

Keyword(s):

Membrane Protein ◽

Single Molecule ◽

Ionic Current ◽

Md Simulations ◽

Energy Spectra ◽

Membrane Channels ◽

Non Covalent Interactions ◽

Protein Channels ◽

Covalent Interactions ◽

Molecule Interaction

Millions of years of evolution have produced membrane protein channels capable of efficiently moving ions across the cell membrane. The underlying fundamental mechanisms that facilitate these actions greatly contribute to the weak non-covalent interactions. However, uncovering these dynamic interactions and its synergic network effects still remains challenging in both experimental techniques and molecule dynamics (MD) simulations. Here, we present a rational strategy that combines MD simulations and frequency-energy spectroscopy to identify and quantify the role of non-covalent interactions in carrier transport through membrane protein channels, as encoded in traditional single channel recording or ionic current. We employed wild-type aerolysin transporting of methylcytosine and cytosine as a model to explore the dynamic ionic signatures with non-stationary and non-linear frequency analysis. Our data illuminate that methylcytosine experiences strong non-covalent interactions with the aerolysin nanopore at Region 1 around R220 than cytosine, which produces characteristic frequency-energy spectra. Furthermore, we experimentally validate the obtained hypothesis from frequency-energy spectra by designing single-site mutation of K238G which creates significantly enhanced non-covalent interactions for the recognition of methylcytosine. The frequency-energy spectrum of ions flowing inside membrane channels constitutes a single-molecule interaction spectrum, which bridges the gap between traditional ionic current recording and the MD simulations, facilitating the qualitative and quantitive description of the non-covalent interactions inside membrane channels.

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