Kinetics of pHLIP peptide insertion into and exit from a membrane

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.

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Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Molecular Endocrinology ◽

10.1210/me.2011-1160 ◽

2011 ◽

Vol 25 (10) ◽

pp. 1804-1818 ◽

Cited By ~ 37

Author(s):

K. Coopman ◽

R. Wallis ◽

G. Robb ◽

A. J. H. Brown ◽

G. F. Wilkinson ◽

...

Keyword(s):

Ligand Binding ◽

Structural Model ◽

Transmembrane Domain ◽

Transmembrane Helix ◽

Receptor Activation ◽

Agonist Affinity ◽

Camp Generation ◽

N Terminus ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

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Tryptophan Residues Flanking the Second Transmembrane Helix (TM2) Set the Signaling State of the Tar Chemoreceptor†

Biochemistry ◽

10.1021/bi048969d ◽

2005 ◽

Vol 44 (4) ◽

pp. 1268-1277 ◽

Cited By ~ 47

Author(s):

Roger R. Draheim ◽

Arjan F. Bormans ◽

Run-zhi Lai ◽

Michael D. Manson

Keyword(s):

Transmembrane Helix ◽

Tryptophan Residues

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Influence of low vacuum levels on milking characteristics of sheep, goat and buffalo

Journal of Agricultural Engineering ◽

10.4081/jae.2013.285 ◽

2013 ◽

Vol 44 (2s) ◽

Cited By ~ 3

Author(s):

Maria Caria ◽

Carlo Boselli ◽

Lelia Murgia ◽

Remo Rosati ◽

Antonio Pazzona

Keyword(s):

High Vacuum ◽

Vacuum Level ◽

Machine Milking ◽

The World ◽

Study Characteristics ◽

Principal Factors ◽

Low Levels ◽

Kinetics Of ◽

Teat Canal ◽

Different Parts

Different settings of the operating parameters (pulsator rate, pulsator ratio and vacuum) are used for milking dairy species in different parts of the world. The level of the operating vacuum in machine milking is one of the principal factors which influence the integrity of the tissues and the milk quality. High vacuum levels (>42 kPa) are often used to facilitate the opening of the teat canal by overcoming the biological closing forces whithin the teat sphincter, but can result in severe machine-induced teat tissue damage. In this study characteristics and performances of mechanical milking at low vacuum levels have been investigated in different dairy species. Milking times and milk productions have been obtained from milk emission curves, recorded by electronic milk-meters (LactoCorder®) during the milking at different vacuum levels of sheep, goats and buffaloes. The results of the comparative experiments clearly indicate that a low vacuum level modifies the kinetics of milk emission, the machine-on time and, thus, the throughput of milking system, in all the dairy species considered. Milk yield was satisfactory at any level tested, showing that low vacuums can be adequate to completely empty the udder. Slight differences were found across species concerning the increase in the milking time per head associated with low levels of milking vacuum Our study represents a contribution to encourage the decrease of the working vacuum during mechanical milking, also for those dairy species generally considered hard to be milked, as buffaloes. Milking should be performed applying the lowest vacuum level, compatible with not excessively prolonging milking time, in line with the animal welfare on dairy husbandry.

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Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

Biochemical Journal ◽

10.1042/bj20071436 ◽

2008 ◽

Vol 411 (3) ◽

pp. 523-530 ◽

Cited By ~ 12

Author(s):

Gary S. Laco ◽

Yves Pommier

Keyword(s):

Amino Acids ◽

Topoisomerase I ◽

Electrostatic Interactions ◽

Functional Domain ◽

Site Mutation ◽

Supercoiled Dna ◽

Terminal Domain ◽

Tryptophan Residues ◽

N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

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Functional analysis of the polar amino acid in TatA transmembrane helix

10.1101/2021.11.30.470661 ◽

2021 ◽

Author(s):

Binhan Hao ◽

Wenjie Zhou ◽

Steven M Theg

Keyword(s):

Amino Acid ◽

Potential Role ◽

Transmembrane Helix ◽

Amino Acid Side Chain ◽

Polar Amino Acid ◽

Twin Arginine Translocation ◽

N Terminus ◽

Biochemical Approach ◽

Folded Proteins ◽

Highly Correlated

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (PMF) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different PMF components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

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A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909722116 ◽

2019 ◽

Vol 116 (38) ◽

pp. 18822-18826 ◽

Cited By ~ 2

Author(s):

Runze Li ◽

Dinesh Dhankhar ◽

Jie Chen ◽

Thomas C. Cesario ◽

Peter M. Rentzepis

Keyword(s):

Fluorescence Intensity ◽

Protein Unfolding ◽

Uv Light ◽

Quantum Yields ◽

Continuous Irradiation ◽

Minute Period ◽

Bacteria Inactivation ◽

Tryptophan Residues ◽

Inactivation Mechanism ◽

Kinetics Of

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

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