Properties of integral membrane protein structures: Derivation of an implicit membrane potential

Ulmschneider, Martin B.; Sansom, Mark S. P.; Nola, Alfredo Di

doi:10.1002/prot.20334

Cited by 181 publications

(241 citation statements)

References 112 publications

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“…The mouth of this tunnel is surrounded by three groups of residues (196-202, 234-236, and 261-271) that are likely to integrate into the lipid bilayer owing to their overall hydrophobicity. Several aromatic residues are found in these segments, suggesting that the depth of RPE65 membrane interaction is restricted to the most proximal portions of phospholipid acyl chains with respect to the polar head groups (24). A hydrophobic interaction with the membrane is consistent with the observation that detergent is required for quantitative solubilization of RPE65.…”

Section: Resultssupporting

confidence: 75%

Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Kiser

Golczak²,

Lodowski³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

136

227

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Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-Å resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.isomerization ͉ metalloprotein ͉ monotopic membrane protein V ision begins when the 11-cis-retinylidene chromophore of rhodopsin is photoisomerized to all-trans-retinylidene, a process resulting in receptor activation and transduction of the light signal (1). After rhodopsin is photoactivated, it is no longer responsive to light, so for vision to continue, a trans-to-cis isomerization mechanism must be present to regenerate lightsensitive visual pigments. In vertebrates, after photoisomerization, all-trans-retinylidene is hydrolyzed from rhodopsin, reduced to all-trans-retinol, and transported to a tissue adjacent to the photoreceptor layer known as the retinal pigment epithelium (RPE), where enzymatic isomerization occurs (2). An RPEspecific, microsomal membrane protein with an apparent molecular mass of 65 kDa, known as RPE65, was determined to be responsible for trans-to-cis retinoid isomerase activity in the RPE (3-5). The importance of this protein in visual function is also evident from the observation that certain RPE65 mutations cause a form of the hereditary childhood blinding disease known as Leber congenital amaurosis (LCA) or the less severe, lateronset disease, retinitis pigmentosa (RP) (6-8).Based on sequence homology, RPE65 belongs to a family of carotenoid cleavage oxygenase (CCO) enzymes that oxidatively cleave ␤-carotene or apocarotenoids (9-11). However, RPE65 is distinct from all other members of this family in that it simultaneously cleaves and isomerizes all-trans-retinyl esters to 11-cisretinol and a fatty acid rather than oxidatively cleaving carotenoids (3-5, 11, 12). Unlike the reactions catalyzed by other CCO family members, there is no obvious role for molecular oxygen in RPE65 enzymology. The only family member with a known crystal structure is an apocarotenoid oxygenase from Synechocystis that is 25% identical and 42% homologous to human RPE65 (13). All members of this fa...

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Section: Resultssupporting

confidence: 75%

Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Kiser

Golczak²,

Lodowski³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

136

227

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“…The set of TM protein mimetics we designed and prepared are polymers of a peptide with the core sequence NH 2 -SKSKS-Leu 20 -SKSKS-NH 2 , termed "pL 20 " (13). The average length, high hydrophobicity, and abundance of Leu in natural membrane-spanning regions are recapitulated in the 20-Leu segment of pL 20 , whereas its basic Lys and hydrophilic Ser residues resemble the native termini of such sequences (17). Sequence patterns that mediate oligomerization (e.g., refs.…”

Section: Resultsmentioning

confidence: 99%

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Rath

Cunningham

Deber

2013

Proc. Natl. Acad. Sci. U.S.A.

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SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of watersoluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20-30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membranespanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE. gel mobility | protein migration | protein identification | apparent size | immunoblotting L aemmli's system for polyacrylamide gel protein electrophoresis in the presence of the detergent SDS (SDS/PAGE) is one of the most cited methodological papers in life sciences (1). The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, resulting in "overnight" adoption of the technique in biochemistry, cell biology, immunology, and virology (2). Considered "the single most useful analytical tool to study protein molecules" (3), SDS/ PAGE is routinely used for simultaneous determination of protein heterogeneity and molecular weight in applications ranging from diagnosis of hereditary red cell membrane disorders to evaluation of recombinant protein expression and purification procedures. Protein analysis by SDS/PAGE is relatively simple, affordable, and rapid (4): A buffer containing a tracking dye and SDS is added to the sample of interest, the mixture is applied to a polyacrylamide gel, and a potential difference is used to drive the dye and the resulting anionic particle composed of protein and dodecyl sulfate (DS) through the gel. The distance traveled by the protein/DS particle from the top of the gel is then divided by that of the dye to obtain relative migration (R f ), and molecular weight [as relative molecular mass (M r )] determined by comparison of this value with a logarithmic plot derived from the R f s and M r s of reference proteins.Fractionation on SDS/PAGE is contro...

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“…This compares with potential of mean force (PMF) results of ∼1 kcal/mol from a nonhelical hexapeptide containing one Trp. 7 Estimates based on Trp scans in designed translocon inserted peptides 31,32 as well as PMFs derived from statistical analyses of membrane protein structures 61 both show barriers of ∼1 kcal/mol per Trp. PMFs on side-chain analogs give barrier heights of ∼0.5-4 kcal/mol per Trp, 62,63 but none for hydrophobic residues (Ala, Leu), indicating the lack of the polar peptide backbone contributions in these scales.…”

Section: Discussionmentioning

confidence: 99%

Mechanism and Kinetics of Peptide Partitioning into Membranes from All-Atom Simulations of Thermostable Peptides

et al. 2010

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Partitioning properties of transmembrane (TM) polypeptide segments directly determine membrane protein folding, stability, and function, and their understanding is vital for rational design of membrane active peptides. However, direct determination of water-to-bilayer transfer of TM peptides has proved difficult. Experimentally, sufficiently hydrophobic peptides tend to aggregate, while atomistic computer simulations at physiological temperatures cannot yet reach the long time scales required to capture partitioning. Elevating temperatures to accelerate the dynamics has been avoided, as this was thought to lead to rapid denaturing. However, we show here that model TM peptides (WALP) are exceptionally thermostable. Circular dichroism experiments reveal that the peptides remain inserted into the lipid bilayer and are fully helical, even at 90°C. At these temperatures, sampling is ∼50-500 times faster, sufficient to directly simulate spontaneous partitioning at atomic resolution. A folded insertion pathway is observed, consistent with three-stage partitioning theory. Elevated temperature simulation ensembles further allow the direct calculation of the insertion kinetics, which is found to be first-order for all systems. Insertion barriers are ∆H in q ) 15 kcal/mol for a general hydrophobic peptide and ∼23 kcal/mol for the tryptophan-flanked WALP peptides. The corresponding insertion times at room temperature range from 8.5 µs to 163 ms. High-temperature simulations of experimentally validated thermostable systems suggest a new avenue for systematic exploration of peptide partitioning properties.

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Properties of integral membrane protein structures: Derivation of an implicit membrane potential

Cited by 181 publications

References 112 publications

Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Mechanism and Kinetics of Peptide Partitioning into Membranes from All-Atom Simulations of Thermostable Peptides

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