Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments.

Huang, Kuo‐Sen; Bayley, Hagan; Ming-yang, Liao; London, Erwin; Khorana, H G

doi:10.1016/s0021-9258(19)69526-8

Cited by 342 publications

(124 citation statements)

References 56 publications

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“…CD spectra indicate that in the D-state, BR retains~50% of the initial helicity (2). In a separate study, we concluded from DEER distance measurements of labels at the ends of each helix that in the D-state most helices retain some of their N-state secondary structure despite disruption of the helices (V. Krishnamani and J. K. Lanyi, unpublished).…”

Section: Discussionmentioning

confidence: 93%

“…To increase the signal/noise ratio of the ESR spectrum, we made the initial concentration of the denatured samples 10-fold greater (100 mM) than that used in previous refolding experiments (2,3,7,10,14,19). The purple membrane was solubilized by addition of SDS in pH 6.5 phosphate buffer to maintain the 1:400 SDS/protein molar ratio of the previously established denaturing conditions (2,7). We regenerated the partially denatured BR in SDS micelles by adding an equal volume of DMPC/CHAPS micelle suspension (pH 6.5) as described elsewhere (7).…”

Section: In Vitro Refoldingmentioning

confidence: 99%

“…The diffraction structure of BR at 1.55 Å resolution revealed a trimer with each monomer consisting of a hepta-helical transmembrane bundle (16), and the structural basis of its function as a light-driven proton pump (17). The seven helices enclose a retinal whose photoisomerization from all-trans to 13-cis triggers the first step of the light-dependent proton transport (2). Here, we report on the kinetics of the refolding of the helices and the association of pairs of helices of BR, starting from the partially denatured state present in SDS micelles.…”

Section: Introductionmentioning

confidence: 99%

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Structural Changes in Bacteriorhodopsin during In Vitro Refolding from a Partially Denatured State

Krishnamani

Lanyi

2011

Biophysical Journal

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We report on the formation of the secondary and tertiary structure of bacteriorhodopsin during its in vitro refolding from an SDS-denatured state. We used the mobility of single spin labels in seven samples, attached at various locations to six of the seven helical segments to engineered cysteine residues, to follow coil-to-helix formation. Distance measurements obtained by spin dipolar quenching in six samples labeled at either the cytoplasmic or extracellular ends of pairs of helices revealed the time dependence of the recovery of the transmembrane helical bundle. The secondary structure in the majority of the helical segments refolds with a time constant of <100-140 ms. Recovery of the tertiary structure is achieved by sequential association of the helices and occurs in at least three distinct steps with time constants of 1), well below 1 s; 2), 3-4 s; and 3), 60-130 s (the latter depending on the helical pair). The slowest of these processes occurs in concert with recovery of the retinal chromophore.

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Section: Discussionmentioning

confidence: 93%

Section: In Vitro Refoldingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structural Changes in Bacteriorhodopsin during In Vitro Refolding from a Partially Denatured State

Krishnamani

Lanyi

2011

Biophysical Journal

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“…Protein expression, purification and spectroscopic characterization of the functional proton pump from Exiguobacterium sibiricum (ESR) was performed as described previously [23]. Reconstitution of ESR (from DDM) and formation of phospholipid proteoliposomes was carried out by cholate dialysis [47]. The protein to lipid molar ratio was 1:1700.…”

Section: Proton Transport Measurementsmentioning

confidence: 99%

Structure Elucidation and Functional Studies of a Novel β-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta

et al. 2020

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Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a β-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known β-hairpin AMP family—tachyplesins and polyphemusins from the horseshoe crabs. The β-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted β-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known β-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.

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“…In 1977 Gobind Khorana's laboratory reported that bR remained intact after solubilization in detergent and complete delipidation [7]. Subsequently, Khorana and coworkers showed that the purple protein-retinal complex, isolated from detergent-solubilized purple membrane, could be completely denatured and refolded [8]. Khorana and Ovchinnikov individually determined the polypeptide sequence, thus defining another landmark [9,10].…”

Section: Br As a Paradigmmentioning

confidence: 99%

It's not just a phase: crystallization and X-ray structure determination of bacteriorhodopsin in lipidic cubic phases

Gouaux

1998

Structure

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Utilization of lipidic cubic phases in the crystallization of bacteriorhodopsin (bR) has yielded long sought after crystals that diffract X-rays to 2 A resolution. The resulting structure provides new information on the protein conformation and the mechanism of proton translocation. Crystallization of bR via lipidic cubic phases may be a harbinger of new membrane protein crystallization strategies.

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Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments.

Cited by 342 publications

References 56 publications

Structural Changes in Bacteriorhodopsin during In Vitro Refolding from a Partially Denatured State

Structural Changes in Bacteriorhodopsin during In Vitro Refolding from a Partially Denatured State

Structure Elucidation and Functional Studies of a Novel β-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta

It's not just a phase: crystallization and X-ray structure determination of bacteriorhodopsin in lipidic cubic phases

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