Structural Biology and Structure–Function Relationships of Membrane Proteins

Abstract

To understand the biological complexity of life, one needs to investigate how biomolecules behave and interact with each other at a molecular level [...]

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells

“…This study found significant difference in final protein yields between the two protocols, with the protein yield from K562 higher when utilizing Protocol 2, compared with protein concentration yield when using Protocol 1. This current adapted protocol is supported by previous investigations that reported wide variations in protein yield and reproducibility by existing protein extraction methods, which has been widely reported as a big challenge in membrane proteomic studies (Alberts et al, 2002;Bünger et al, 2009;Grabski, 2009;Lin and Guidotti, 2009;Janson, 2012;Lai, 2013;Scheiter et al, 2013;Kubicek et al, 2014;Grant, 2016;Lee, 2017;Reis and Moraes, 2019;Birch et al, 2020;Moraes and Quigley, 2021). No one cell disruption approach is better since each of these approaches has limitations and challenges.…”

“…Membrane protein isolation is a multiple steps process, and various approaches are used to disrupt cell membranes including mechanical-based techniques such as sonication, French press and grinding as well as nonmechanical-based approaches such as osmotic shock, freeze thawing and detergent-based approaches (Lin and Guidotti, 2009;Janson, 2012). Cell lysis liberates various cell contaminating molecular species, inevitably disrupting, and potentially compromising the structural and functional integrity of membrane proteins (Birch et al, 2020;Moraes and Quigley, 2021). Removal of interfering substances can be achieved by various techniques including acid, salt, and alcohol precipitation methods, usually combined with differential centrifugation (Janson, 2012;Birch et al, 2020;Moraes and Quigley, 2021).…”

“…Cell lysis liberates various cell contaminating molecular species, inevitably disrupting, and potentially compromising the structural and functional integrity of membrane proteins (Birch et al, 2020;Moraes and Quigley, 2021). Removal of interfering substances can be achieved by various techniques including acid, salt, and alcohol precipitation methods, usually combined with differential centrifugation (Janson, 2012;Birch et al, 2020;Moraes and Quigley, 2021). Therefore, to evaluate the best approach for lysing NK cells and precipitate membrane proteins, a comparison of two methods which employed different protein extraction strategies was performed.…”

“…Membrane proteins perform essential and diverse roles in various cellular processes such as catalysis of chemical reactions, movement of ions across membranes, and transmission of signals between cells and their environments (Lin and Guidotti, 2009;Birch et al, 2020). Mutations and misfolding of membrane proteins are associated with various disorders, including neurological indications, the perception of pain, cardiac disorders, kidney failure and blindness (Bagal et al, 2013;Moraes and Quigley, 2021). Given membrane proteins' diverse tissue distribution and membrane localization, they provide potential targets in proteomic studies, drug discovery and disease management (Lin and Guidotti, 2009;Birch et al, 2020;Moraes and Quigley, 2021).…”

“…Mutations and misfolding of membrane proteins are associated with various disorders, including neurological indications, the perception of pain, cardiac disorders, kidney failure and blindness (Bagal et al, 2013;Moraes and Quigley, 2021). Given membrane proteins' diverse tissue distribution and membrane localization, they provide potential targets in proteomic studies, drug discovery and disease management (Lin and Guidotti, 2009;Birch et al, 2020;Moraes and Quigley, 2021). However, membrane protein expression concentrations are low in their native sources.…”