scholarly journals Intentional crystal-contact-free space in protein crystal

2014 ◽  
Vol 70 (a1) ◽  
pp. C339-C339
Author(s):  
Rei Matsuoka ◽  
Yasuaki Komuro ◽  
Yuji Sugita ◽  
Daisuke Kohda
Keyword(s):  
Electron Density ◽  
Free Space ◽  
Protein Import ◽  
Model Building ◽  
Protein Complexes ◽  
Dynamics Simulation ◽  
Protein Crystal ◽  
Α Helix ◽  
Contact Free ◽  
Crystal Contact

To understand the function of proteins, it is essential to perform the structural analysis of the protein complexes with ligands, such as substrates or partner molecules. The motions of ligands are restricted by the contacts with neighbor protein molecules in the crystal lattice. Here, we propose a new technique to analyze dynamics of a ligand in the bound state preserved in the crystal-contact-free space, which is intentionally created in protein crystals. We used Tom20 as a target protein. Tom20 functions as a general protein import receptor, by recognizing N-terminal signal sequences (presequences) of mitochondrial matrix proteins. Our working hypothesis is that the promiscuous specificity of Tom20 is attributed to the large mobility of the presequneces in the binding groove of Tom20 (1,2). Our aim is to obtain electron density that reflects the large mobility of a presequence in the crystal-contact-free space. In order to create the crystal-contact-free space, we took advantage of a protein fused with maltose binding protein (MBP). The key of the design is the connection of the two proteins firmly. We fused the C-terminal α-helix of MBP and the N-terminal α-helix of Tom20 seamlessly. After a systematic model building study, we decided to use a design with four residues inserted in the linker region. We found smeared electron density in the binding site of presequences in the difference Fourier electron-density map. We attached an iodine atom at the N-terminus of the presequence and confirmed the N-terminal position in the smeared electron density. We performed molecular dynamics simulation without the tethering in solution (3). The electron density simulated from the MD trajectory was fully consistent with the smeared electron density in the crystal contact-free space. We concluded that the smeared electron density corresponded to the partially overlapping region of the multiple states of the bound presequence.

scholarly journals Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study

2020 ◽  
Vol 1864 (2) ◽  
pp. 129418 ◽  
Author(s):  
Siqin Bala ◽  
Shoko Shinya ◽  
Arpita Srivastava ◽  
Marie Ishikawa ◽  
Atsushi Shimada ◽  
...  
Keyword(s):  
Protein Crystal ◽  
Flexible Loop ◽  
Contact Free ◽  
Crystal Contact

scholarly journals Rational design of crystal contact-free space in protein crystals for analyzing spatial distribution of motions within protein molecules

2016 ◽  
Vol 25 (3) ◽  
pp. 754-768 ◽  
Author(s):  
Rei Matsuoka ◽  
Atsushi Shimada ◽  
Yasuaki Komuro ◽  
Yuji Sugita ◽  
Daisuke Kohda
Keyword(s):  
Spatial Distribution ◽  
Free Space ◽  
Rational Design ◽  
Protein Crystals ◽  
Protein Molecules ◽  
Contact Free ◽  
Crystal Contact

scholarly journals Protein-complex structure completion usingIPCAS(Iterative Protein Crystal structure Automatic Solution)

2015 ◽  
Vol 71 (7) ◽  
pp. 1487-1492 ◽  
Author(s):  
Weizhe Zhang ◽  
Hongmin Zhang ◽  
Tao Zhang ◽  
Haifu Fan ◽  
Quan Hao
Keyword(s):  
Crystal Structure ◽  
Protein Complex ◽  
Model Building ◽  
Protein Complexes ◽  
Direct Method ◽  
Complex Structure ◽  
Protein Crystal ◽  
Molecular Replacement ◽  
Protein Crystal Structure ◽  
Building Program

Protein complexes are essential components in many cellular processes. In this study, a procedure to determine the protein-complex structure from a partial molecular-replacement (MR) solution is demonstrated using a direct-method-aided dual-space iterative phasing and model-building program suite,IPCAS(Iterative Protein Crystal structure Automatic Solution). TheIPCASiteration procedure involves (i) real-space model building and refinement, (ii) direct-method-aided reciprocal-space phase refinement and (iii) phase improvement through density modification. The procedure has been tested with four protein complexes, including two previously unknown structures. It was possible to useIPCASto build the whole complex structure from one or less than one subunit once the molecular-replacement method was able to give a partial solution. In the most challenging case,IPCASwas able to extend to the full length starting from less than 30% of the complex structure, while conventional model-building procedures were unsuccessful.

Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

1992 ◽  
Vol 25 (2) ◽  
pp. 205-210 ◽  
Author(s):  
L. J. Keefe ◽  
E. E. Lattman ◽  
C. Wolkow ◽  
A. Woods ◽  
M. Chevrier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Electron Density ◽  
Mass Spectrometric ◽  
Amino Acid Sequences ◽  
Data Matrix ◽  
Protein Crystal ◽  
Insertion Mutant ◽  
Laser Desorption Mass Spectrometry ◽  
X Ray

Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 Å resolution and refined to a crystallographic R value of 0.170 [Keefe & Lattman (1992). In preparation]. A single residue has been inserted in the C-terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature.

scholarly journals Rational thermostabilisation of four-helix bundle dimeric de novo proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shin Irumagawa ◽  
Kaito Kobayashi ◽  
Yutaka Saito ◽  
Takeshi Miyata ◽  
Mitsuo Umetsu ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Protein Complexes ◽  
Salt Bridge ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Helix Bundle ◽  
Stable Mutant ◽  
Four Helix Bundle ◽  
The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

Assessing the ligand selectivity of sphingosine kinases using molecular dynamics and MM-PBSA binding free energy calculations

2016 ◽  
Vol 12 (4) ◽  
pp. 1174-1182 ◽  
Author(s):  
Liang Fang ◽  
Xiaojian Wang ◽  
Meiyang Xi ◽  
Tianqi Liu ◽  
Dali Yin
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Model Building ◽  
Binding Free Energy ◽  
Homology Model ◽  
Free Energy Calculations ◽  
Dynamics Simulation ◽  
Ligand Selectivity ◽  
Sphingosine Kinases ◽  
Binding Free Energy Calculations

Three residues of SK1 were identified important for selective SK1 inhibitory activity via SK2 homology model building, molecular dynamics simulation, and MM-PBSA studies.

scholarly journals Computational Prediction of Heteromeric Protein Complex Disassembly Order with Hybrid Monte Carlo/Molecular Dynamics Simulation

2022 ◽  
Author(s):  
Ikuo Kurisaki ◽  
Shigenori Tanaka
Keyword(s):  
Molecular Dynamics ◽  
Monte Carlo ◽  
Protein Complex ◽  
Protein Complexes ◽  
Structural Bioinformatics ◽  
Dynamics Simulation ◽  
Tryptophan Synthase ◽  
Selection Scheme ◽  
Hybrid Monte Carlo ◽  
Multimeric Protein

The physicochemical entity of biological phenomenon in the cell is a network of biochemical reactions and the activity of such a network is regulated by multimeric protein complexes. Mass spectroscopy (MS) experiments and multimeric protein docking simulations based on structural bioinformatics techniques have revealed the molecular-level stoichiometry and static configuration of subcomplexes in their bound forms, then revealing the subcomplex populations and formation orders. Meanwhile, these methodologies are not designed to straightforwardly examine temporal dynamics of multimeric protein assembly and disassembly, essential physicochemical properties to understand functional expression mechanisms of proteins in the biological environment. To address the problem, we had developed an atomistic simulation in the framework of the hybrid Monte Carlo/Molecular Dynamics (hMC/MD) method and succeeded in observing disassembly of homomeric pentamer of the serum amyloid P component protein in experimentally consistent order. In this study, we improved the hMC/MD method to examine disassembly processes of the tryptophan synthase tetramer, a paradigmatic heteromeric protein complex in MS studies. We employed the likelihood-based selection scheme to determine a dissociation-prone subunit pair at each hMC/MD simulation cycle and achieved highly reliable predictions of the disassembly orders with the success rate over 0.9 without a priori knowledge of the MS experiments and structural bioinformatics simulations. We similarly succeeded in reliable predictions for the other three tetrameric protein complexes. These achievements indicate the potential availability of our hMC/MD approach as the general purpose methodology to obtain microscopic and physicochemical insights into multimeric protein complex formation.

scholarly journals Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca

2021 ◽  
Author(s):  
Patrick Brendan Timmons ◽  
Chandralal M Hewage
Keyword(s):  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Dynamics Simulation ◽  
Peptide Sequence ◽  
Helical Conformation ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Anticancer Activities ◽  
American Bullfrog ◽  
Α Helix

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defense peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture. The structure is defined by an α-helix that spans between Ile6-Ala26, and a cyclic disulphide bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulphate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its α-helical conformation between residues Ile6-Ala26, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

Sign in / Sign up

Export Citation Format

Share Document