Molecular architecture of a membrane-spanning hormone acyltransferase required for metabolic regulation

Campana, M.; Irudayanathan, Flaviyan Jerome; Davis, Tasha R.; McGovern-Gooch, Kayleigh R.; Loftus, Rosemary; Ashkar, Mohammad; Escoffery, Najae; Navarro, Melissa; Sieburg, Michelle; Nangia, Shikha; Hougland, James L.

doi:10.1101/556233

Cited by 2 publications

(4 citation statements)

References 32 publications

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“…Details regarding generation of the computational model for human GOAT structure were discussed in our previous work. 17 These systems were subjected to a series of energy minimization and equilibration steps with the input files generated from CHARMM-GUI solution builder. 59,[61][62] The CHARMM36m force field parameters were used for GOAT protein, lipids, detergents, salt (0.15M NaCl), and explicit TIP3P water.…”

Section: Methodsmentioning

confidence: 99%

“…The generation of the hGOAT structure computational model was described in our previous work. 17 Following energy minimization and equilibration, a 200 ns atomistic molecular dynamics run was performed, and protein stability was determined by the average root mean square fluctuation (RMSF) of hGOAT during the simulation (Figure 2A). Analysis of average ΔRMSF GOAT structural stabilization by detergents relative to membrane-equilibrated structure yielded a distinct rank for the eight examined, with FOS-12, CHAPS, and LMNG supporting the lowest ΔRMSF (Figure 2B).…”

Section: Computational Screening Of Hgoat Detergent Solubilizationmentioning

confidence: 99%

“…[15][16] Application of metagenomics and coevolutionary contact analysis provide additional pairwise distance constraints for structural modeling. [17][18][19] Most recently, machine learning and artificial intelligencebased tools such as Alphafold and its progeny have led to an explosion in predicted protein structures covering many organismal proteomes. 20 While these modeling approaches offer important information regarding the structures of unsolved proteins, they cannot offer the resolution of experimentally determined protein structures.…”

Section: Introductionmentioning

confidence: 99%

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A combined computational-biochemical approach offers an accelerated path to membrane protein solubilization

Pierce

Novak

et al. 2023

Preprint

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Membrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to solubilize these proteins, with this approach requiring a careful balance between protein solubilization and denaturation. Determining which detergent is most appropriate for a given protein has largely been done empirically through screening, which requires large amounts of membrane protein and associated resources. Here we describe an alternative to conventional detergent screening using a computational modeling approach to identify the most likely candidate detergents for solubilizing a protein of interest. We demonstrate our approach using ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase (MBOAT) family of integral membrane enzymes that has not been solubilized or purified in active form. A computationally derived GOAT structural model provides the only structural information required for this approach. Using computational analysis of detergent ability to penetrate phospholipid bilayers and stabilize the GOAT structure, a panel of common detergents were rank ordered for their proposed ability to solubilize GOAT. Independently, we biologically screened these detergents for their solubilization of fluorescently tagged GOAT constructs. We found computational prediction of protein structural stabilization was the better predictor of detergent solubilization ability, but neither approach was effective for predicting detergents that would support GOAT enzymatic function. The current rapid expansion of membrane protein computational models lacking experimental structural information and our computational detergent screening approach can greatly improve the efficiency of membrane protein detergent solubilization supporting downstream functional and structural studies.

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Section: Methodsmentioning

confidence: 99%

Section: Computational Screening Of Hgoat Detergent Solubilizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A combined computational-biochemical approach offers an accelerated path to membrane protein solubilization

Pierce

Novak

et al. 2023

Preprint

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“…Given the fact that available ab initio models were inconsistent with other data we constructed our own models of Mt2055 with DMPfold. The inclusion of metagenomic sequence data 26,67,68 was used to generate a large MSA lifting Neff value 69 from 1648 when using Uniref90 35 to 7470 (Supplementary Figure 4). The resulting output model (Fig.…”

Section: Dmpfold Modelling Of the Archaea Mt2055 Protein Constructs Amentioning

confidence: 99%

In silico prediction of structure and function for a large family of transmembrane proteins that includes human Tmem41b

Mesdaghi

Murphy

Rodríguez

et al. 2020

Preprint

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Recent strides in computational structural biology have opened up an opportunity to understand previously mysterious uncharacterised proteins. The under-representation of transmembrane proteins in the Protein Data Bank highlights the need to apply new and advanced bioinformatics methods to shed light on their structure and function. This study focuses on such a family; transmembrane proteins containing the Pfam domain PF09335 ('SNARE_ASSOC'/ 'VTT '/'Tvp38'). One prominent member, Tmem41b, has been shown to be involved in early stages of autophagosome formation and is vital in mouse embryonic development. Here we use evolutionary covariance-derived information not only to construct and validate ab initio models but also to make domain boundary predictions and infer local structural features. The results from the structural bioinformatics analysis of Tmem41b and its homologues show that they contain a tandem repeat that is clearly visible in evolutionary covariance data but much less so by sequence analysis. Furthermore, crossreferencing of other prediction data with the covariance analysis shows that the internal repeat features 2-fold rotational symmetry. Ab initio modelling of Tmem41b reinforces these structural predictions. Local structural features predicted to be present in Tmem41b are also present in Cl -/H + antiporters. These results together strongly point to Tmem41b and its homologues as being transporters for an as-yet uncharacterised substrate and possibly using H + antiporter activity as its mechanism for transport.

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Molecular architecture of a membrane-spanning hormone acyltransferase required for metabolic regulation

Cited by 2 publications

References 32 publications

A combined computational-biochemical approach offers an accelerated path to membrane protein solubilization

A combined computational-biochemical approach offers an accelerated path to membrane protein solubilization

In silico prediction of structure and function for a large family of transmembrane proteins that includes human Tmem41b

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