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1.
Anal Chem ; 96(42): 16768-16776, 2024 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-39394983

RESUMEN

Native mass spectrometry (MS) reveals the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer. However, detergents commonly used for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility in characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable the opportunity to characterize lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find that protein-lipid interactions not only are protein-dependent but also can be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl-N,N-dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery as well as biochemical and structural investigations.


Asunto(s)
Detergentes , Proteínas de Escherichia coli , Espectrometría de Masas , Detergentes/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Acuaporinas/química , Acuaporinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Metilaminas/química , Dimetilaminas/química , Proteínas de Transporte de Catión
2.
bioRxiv ; 2024 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-38979331

RESUMEN

Native mass spectrometry (MS) is revealing the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer; however, commonly used detergents for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility for characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable characterization of lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find protein-lipid interactions are not only protein-dependent but can also be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl-N,N-dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery, as well as biochemical and structural investigations.

3.
J Am Soc Mass Spectrom ; 35(7): 1516-1522, 2024 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-38843438

RESUMEN

TREK2, a two-pore domain potassium channel, is recognized for its regulation by various stimuli, including lipids. While previous members of the TREK subfamily, TREK1 and TRAAK, have been investigated to elucidate their lipid affinity and selectivity, TREK2 has not been similarly studied in this regard. Our findings indicate that while TRAAK and TREK2 exhibit similarities in terms of electrostatics and share an overall structural resemblance, there are notable distinctions in their interaction with lipids. Specifically, SAPI(4,5)P2,1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate) exhibits a strong affinity for TREK2, surpassing that of dOPI(4,5)P2,1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate), which differs in its acyl chains. TREK2 displays lipid binding preferences not only for the headgroup of lipids but also toward the acyl chains. Functional studies draw a correlation for lipid binding affinity and activity of the channel. These findings provide important insight into elucidating the molecular prerequisites for specific lipid binding to TREK2 important for function.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Humanos , Unión Proteica , Espectrometría de Masas/métodos , Animales , Modelos Moleculares , Electricidad Estática , Canales de Potasio
4.
J Struct Biol X ; 9: 100097, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38361954

RESUMEN

Aquaporin Z (AqpZ), a bacterial water channel, forms a tetrameric complex and, like many other membrane proteins, activity is regulated by lipids. Various methods have been developed to facilitate structure determination of membrane proteins, such as the use of antibodies. Here, we graft onto AqpZ the ALFA tag (AqpZ-ALFA), an alpha helical epitope, to make use of the high-affinity anti-ALFA nanobody (nB). Native mass spectrometry reveals the AqpZ-ALFA fusion forms a stable, 1:1 complex with nB. Single-particle cryogenic electron microscopy studies reveal the octameric (AqpZ-ALFA)4(nB)4 complex forms a dimeric assembly and the structure was determined to 1.9 Å resolution. Dimerization of the octamer is mediated through stacking of the symmetrically bound nBs. Tube-like density is also observed, revealing a potential cardiolipin binding site. Grafting of the ALFA tag, or other epitope, along with binding and association of nBs to promote larger complexes will have applications in structural studies and protein engineering.

5.
Chem Sci ; 14(48): 14243-14255, 2023 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-38098719

RESUMEN

Cellular membranes are critical to the function of membrane proteins, whether they are associated (peripheral) or embedded (integral) within the bilayer. While detergents have contributed to our understanding of membrane protein structure and function, there remains challenges in characterizing protein-lipid interactions within the context of an intact membrane. Here, we developed a method to prepare proteoliposomes for native mass spectrometry (MS) studies. We first use native MS to detect the encapsulation of soluble proteins within liposomes. We then find the peripheral Gß1γ2 complex associated with the membrane can be ejected and analyzed using native MS. Four different integral membrane proteins (AmtB, AqpZ, TRAAK, and TREK2), all of which have previously been characterized in detergent, eject from the proteoliposomes as intact complexes bound to lipids that have been shown to tightly associate in detergent, drawing a correlation between the two approaches. We also show the utility of more complex lipid environments, such as a brain polar lipid extract, and show TRAAK ejects from liposomes of this extract bound to lipids. These findings underscore the capability to eject protein complexes from membranes bound to both lipids and metal ions, and this approach will be instrumental in the identification of key protein-lipid interactions.

6.
Nat Commun ; 14(1): 5676, 2023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37709761

RESUMEN

Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.


Asunto(s)
Proteínas de la Membrana , Poliaminas , Detergentes , Espermina , Espectrometría de Masas
7.
Anal Chem ; 94(40): 13906-13912, 2022 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-36170465

RESUMEN

Integral membrane proteins are embedded in the biological membrane, where they carry out numerous biological processes. Although lipids present in the membrane are crucial for membrane protein function, it remains difficult to characterize many lipid binding events to membrane proteins, such as those that form the annular belt. Here, we use native mass spectrometry along with the charge-reducing properties of trimethylamine N-oxide (TMAO) to characterize a large number of lipid binding events to the bacterial ammonia channel (AmtB). In the absence of TMAO, significant peak overlap between neighboring charge states is observed, resulting in erroneous abundances for different molecular species. With the addition of TMAO, the weighted average charge state (Zavg) was decreased. In addition, the increased spacing between nearby charge states enabled a higher number of lipid binding species to be observed while minimizing mass spectral peak overlap. These conditions helped us to determine the equilibrium binding constants (Kd) for up to 16 lipid binding events. The binding constants for the first few lipid binding events display the highest affinity, whereas the binding constants for higher lipid binding events converge to a similar value. These findings suggest a transition from nonannular to annular lipid binding to AmtB.


Asunto(s)
Amoníaco , Proteínas de la Membrana , Lípidos/química , Proteínas de la Membrana/química , Metilaminas
8.
J Am Chem Soc ; 144(16): 7048-7053, 2022 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-35421309

RESUMEN

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Cationes Bivalentes/metabolismo , Fosfatidilserinas , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/metabolismo
9.
J Dent Educ ; 70(3): 263-78, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16522755

RESUMEN

Access to oral health care continues to be a problem in the United States. Research has called for innovative approaches to improve access to oral health care and reduce oral health care disparities. Successful alternate approaches have been reported. In 1998 the Kansas Legislature passed a proposal to enhance access to care and manpower needs by allowing dental assistants to provide supragingival scaling, a service traditionally assigned to dental hygienists. In 2000, Mitchell et al. investigated the perceptions of Kansas dental hygienists and scaling dental assistants in relation to House Bill 2724 (HB 2724), which allows dental assistants to perform coronal scaling. The intent of the study was to collect baseline data in relation to HB 2724. The purpose of the present study was to follow up on the impact of HB 2724 six years after legislation. Both groups report satisfaction with their professions: scaling dental assistants believe the delivery of care in Kansas has changed, and areas of Kansas previously noted as dental health professional shortage areas are now served by either a registered dental hygienist or scaling dental assistant.


Asunto(s)
Actitud del Personal de Salud , Asistentes Dentales/estadística & datos numéricos , Higienistas Dentales/psicología , Profilaxis Dental , Accesibilidad a los Servicios de Salud , Asistentes Dentales/legislación & jurisprudencia , Asistentes Dentales/psicología , Higienistas Dentales/legislación & jurisprudencia , Humanos , Kansas , Características de la Residencia , Encuestas y Cuestionarios , Recursos Humanos
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