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Structural Dynamics (Melville, N.Y.) May 2023Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by...
Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by extreme incompleteness and stochastic weighting. In this work, we apply LPSA to an experimental time-resolved serial femtosecond crystallography (TR-SFX) dataset from the membrane protein bacteriorhodopsin (bR) and analyze its parametric sensitivity. While most dynamical modes are contaminated by nonphysical high-frequency features, we identify two dominant modes, which are little affected by spurious frequencies. The dynamics retrieved using these modes shows an isomerization signal compatible with previous findings. We employ synthetic data with increasing timing uncertainty, increasing incompleteness level, pixel-dependent incompleteness, and photon counting errors to investigate the root cause of the high-frequency contamination of our TR-SFX modes. By testing a range of methods, we show that timing errors comparable to the dynamical periods to be retrieved produce a smearing of dynamical features, hampering dynamics retrieval, but with no introduction of spurious components in the solution, when convergence criteria are met. Using model data, we are able to attribute the high-frequency contamination of low-order dynamical modes to the high levels of noise present in the data. Finally, we propose a method to handle missing observations that produces a substantial dynamics retrieval improvement from synthetic data with a significant static component. Reprocessing of the bR TR-SFX data using the improved method yields dynamical movies with strong isomerization signals compatible with previous findings.
PubMed: 37275629
DOI: 10.1063/4.0000178 -
Scientific Reports May 2023In the present study, the dependency of purple membrane (PM) dielectric responses on the wavelength of light in the range 380-750 nm has showed meaningful changes about...
In the present study, the dependency of purple membrane (PM) dielectric responses on the wavelength of light in the range 380-750 nm has showed meaningful changes about the rotation of PM in suspension and about the rotation of bacteriorhodopsin (bR) trimer inside PM, as well. The action spectrum of PM random walk substantiates the existence of two states of bR. One of them (blue edge-state) lies at the blue edge and the other (red edge-state) at the red edge of the visible absorption of bR. The results might bear on correlation of these bands to some bR photocycle intermediates or bR photoproducts. The results implicate the protein-chromophore interactions that eventually underlie protein-lipid interactions. Disrupting the protein-lipid contact during the illumination with light of wavelength in ranges of (410-470 nm) and (610-720 nm) has resulted in emergence of distinct dielectric dispersion at 0.06-0.08 MHz which is comparable to the size of bR trimer or monomer.The work reports on the chromatic adaptation of bR in view of the dielectric spectral parameters of PM. It aimed to explore a correlation seemingly found between the light wavelength and the relaxations of bR trimer inside PM. Changes in rotational diffusion of bR trimer upon blue and red light illumination can influence the three dimensional data storage based on bR, which may implicate bR in bioelectronics.
Topics: Bacteriorhodopsins; Purple Membrane; Action Spectrum; Lipids
PubMed: 37193768
DOI: 10.1038/s41598-023-35121-8 -
Microbiology (Reading, England) Apr 2023is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971...
is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971 discovery of bacteriorhodopsin, the light-driven proton pump, that raised interest in across a range of disciplines, including biophysics, chemistry, molecular evolution and biotechnology. have since contributed to numerous discoveries, such as advances in membrane protein structure determination and the first example of a non-eukaryal glycoprotein. Work on , one of the species used to define Archaea, has also elucidated molecular workings in the third domain. Finally, presents creative solutions to the challenges of life in high salt.
Topics: Halobacterium salinarum; Sodium Chloride; Archaea
PubMed: 37068123
DOI: 10.1099/mic.0.001327 -
Biophysical Reviews Feb 2023The centenary of the birth of H. Gobind Khorana provides an auspicious opportunity to review the origins and evolution of parallel advances in biophysical methodology... (Review)
Review
The centenary of the birth of H. Gobind Khorana provides an auspicious opportunity to review the origins and evolution of parallel advances in biophysical methodology and molecular genetics technology used to study membrane proteins. Interdisciplinary work in the Khorana laboratory in the late 1970s and for the next three decades led to productive collaborations and fostered three subsequent scientific generations whose biophysical work on membrane proteins has led to detailed elucidation of the molecular mechanisms of energy transduction by the light-driven proton pump bacteriorhodopsin (bR) and signal transduction by the G protein-coupled receptor (GPCR) rhodopsin. This review will highlight the origins and advances of biophysical studies of membrane proteins made possible by the application of molecular genetics approaches to engineer site-specific alterations of membrane protein structures.
PubMed: 36909961
DOI: 10.1007/s12551-022-01003-y -
Biophysical Reviews Feb 2023Gobind Khorana's distinguished career spanned nearly six decades (1952-2011). His work resulted in remarkable achievements starting with the complicated synthesis of...
Gobind Khorana's distinguished career spanned nearly six decades (1952-2011). His work resulted in remarkable achievements starting with the complicated synthesis of coenzyme A. He then pioneered the synthesis of DNA oligonucleotides, which enabled him to crack the genetic code. Using this experience, he ventured to accomplish the first complete synthesis of a gene. Not satisfied with elucidating the function of bacteriorhodopsin, Gobind took up another greater challenge, that of spearheading studies on visual rhodopsin, its mechanism of activation, and the consequent signal transduction pathway. This Editorial acts to introduce the articles appearing in this Issue Focus dedicated to celebrating the 100th anniversary of the year of his birth.
PubMed: 36909957
DOI: 10.1007/s12551-023-01045-w -
Biophysical Reviews Feb 2023H.G. Khorana's seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced... (Review)
Review
H.G. Khorana's seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins.
PubMed: 36909952
DOI: 10.1007/s12551-023-01046-9 -
Communications Biology Feb 2023The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures...
The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures have been reported for the K intermediate until now, these differ from each other, especially in terms of the conformation of the retinal chromophore and its interaction with surrounding residues. We report here an accurate X-ray crystallographic analysis of the K structure. The polyene chain of 13-cis retinal is observed to be S-shaped. The side chain of Lys216, which is covalently bound to retinal via the Schiff-base linkage, interacts with residues, Asp85 and Thr89. In addition, the Nζ-H of the protonated Schiff-base linkage interacts with a residue, Asp212 and a water molecule, W402. Based on quantum chemical calculations for this K structure, we examine the stabilizing factors of distorted conformation of retinal and propose a relaxation manner to the next L intermediate.
Topics: Bacteriorhodopsins; Models, Molecular; Proton Pumps; Molecular Conformation; Ion Transport
PubMed: 36808185
DOI: 10.1038/s42003-023-04554-2 -
RSC Advances Feb 2023Rhodopsins, a family of photoreceptive membrane proteins, contain retinal as a chromophore and were firstly identified as reddish pigments from frog retina in 1876.... (Review)
Review
Rhodopsins, a family of photoreceptive membrane proteins, contain retinal as a chromophore and were firstly identified as reddish pigments from frog retina in 1876. Since then, rhodopsin-like proteins have been identified mainly from animal eyes. In 1971, a rhodopsin-like pigment was discovered from the archaeon and named bacteriorhodopsin. While it was believed that rhodopsin- and bacteriorhodopsin-like proteins were expressed only in animal eyes and archaea, respectively, before the 1990s, a variety of rhodopsin-like proteins (called animal rhodopsins or opsins) and bacteriorhodopsin-like proteins (called microbial rhodopsins) have been progressively identified from various tissues of animals and microorganisms, respectively. Here, we comprehensively introduce the research conducted on animal and microbial rhodopsins. Recent analysis has revealed that the two rhodopsin families have common molecular properties, such as the protein structure (, 7-transmembrane structure), retinal structure (, binding ability to - and -retinal), color sensitivity (, UV- and visible-light sensitivities), and photoreaction (, triggering structural changes by light and heat), more than what was expected at the early stages of rhodopsin research. Contrastingly, their molecular functions are distinctively different (, G protein-coupled receptors and photoisomerases for animal rhodopsins and ion transporters and phototaxis sensors for microbial rhodopsins). Therefore, based on their similarities and dissimilarities, we propose that animal and microbial rhodopsins have convergently evolved from their distinctive origins as multi-colored retinal-binding membrane proteins whose activities are regulated by light and heat but independently evolved for different molecular and physiological functions in the cognate organism.
PubMed: 36793294
DOI: 10.1039/d2ra07073a -
International Journal of Molecular... Jan 2023Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane...
Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane-protein interactions had only moderate influences, except for the extraction of the last helix where the membrane-protein interactions had a stronger influence. The up-down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein.
Topics: Bacteriorhodopsins; Molecular Dynamics Simulation; Protein Unfolding; Microscopy, Atomic Force; Protein Denaturation; Protein Folding
PubMed: 36768981
DOI: 10.3390/ijms24032654 -
BMC Bioinformatics Jan 2023Rhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in...
BACKGROUND
Rhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in eukaryotes, bacteria, and archaea. Haloarchaeal rhodopsins are Type-I microbial rhodopsin that elicits various light-driven functions like proton pumping, chloride pumping and Phototaxis behaviour. The industrial application of Ion-pumping Haloarchaeal rhodopsins is limited by the lack of full-length rhodopsin sequence-based classifications, which play an important role in Ion-pumping activity. The well-studied Haloarchaeal rhodopsin is a proton-pumping bacteriorhodopsin that shows promising applications in optogenetics, biosensitized solar cells, security ink, data storage, artificial retinal implant and biohydrogen generation. As a result, a low-cost computational approach is required to identify Ion-pumping Haloarchaeal rhodopsin sequences and its subtype.
RESULTS
This study uses a support vector machine (SVM) technique to identify these ion-pumping Haloarchaeal rhodopsin proteins. The haloarchaeal ion pumping rhodopsins viz., bacteriorhodopsin, halorhodopsin, xanthorhodopsin, sensoryrhodopsin and marine prokaryotic Ion-pumping rhodopsins like actinorhodopsin, proteorhodopsin have been utilized to develop the methods that accurately identified the ion pumping haloarchaeal and other type I microbial rhodopsins. We achieved overall maximum accuracy of 97.78%, 97.84% and 97.60%, respectively, for amino acid composition, dipeptide composition and hybrid approach on tenfold cross validation using SVM. Predictive models for each class of rhodopsin performed equally well on an independent data set. In addition to this, similar results were achieved using another machine learning technique namely random forest. Simultaneously predictive models performed equally well during five-fold cross validation. Apart from this study, we also tested the own, blank, BLAST dataset and annotated whole-genome rhodopsin sequences of PWS haloarchaeal isolates in the developed methods. The developed web server ( https://bioinfo.imtech.res.in/servers/rhodopred ) can identify the Ion Pumping Haloarchaeal rhodopsin proteins and their subtypes. We expect this web tool would be useful for rhodopsin researchers.
CONCLUSION
The overall performance of the developed method results show that it accurately identifies the Ionpumping Haloarchaeal rhodopsin and their subtypes using known and unknown microbial rhodopsin sequences. We expect that this study would be useful for optogenetics, molecular biologists and rhodopsin researchers.
Topics: Bacteria; Bacteriorhodopsins; Light; Protons; Rhodopsin; Rhodopsins, Microbial; Machine Learning
PubMed: 36707759
DOI: 10.1186/s12859-023-05138-x