506
M. Rashid et al.
diphosphate ribose) which are involved in bio-calcification [72]. This area of research
is still open with many more possibilities.
9.3 Membrane Proteins
NMR spectroscopy has been utilized to study proteins, specially where crystallization is difficult such as membrane proteins, intrinsically disordered proteins and
proteins forming fibrils [73–77]. Both solution NMR and ssNMR have been used
to study membrane protein structure, functions and interactions. While purification
membrane proteins are extracted out of the membrane using detergent or similar
molecules, refolded (specially in case of beta barrels proteins) and then reconstituted
in suitable kind of membrane environment for further studies. NMR spectroscopy has
the benefit that it can give both structural and dynamical information of the protein
in native state [74]. The membrane models have evolved with time to make the environment closest to native state. Figure 27 shows some common types of membrane
models which have been used and still being used to prepare membrane proteins,
here adhesion invasion locus (AIL) an outer membrane protein from Yersinia pestis
has been shown inserted inside these membrane models. General membrane models
include, Fig. 27 (a) detergent micelles formed by detergent molecules around protein
where the hydrophobic tail is inside and the headgroup is exposed outside (b) liposomes, which is lipid bilayer without detergent (d and e) nanodiscs are lipid discs
whose sized is controlled by membrane scaffold protein wrapped around the disc
as well as bicelles where the size of the size is controlled by detergent molecules
forming the outer rim of the discs. The structure of the protein taken for example
has been calculated in micelles and nanodiscs [78, 79]. Samples reconstituted in
liposomes, bicelles are studied by ssNMR, while membrane proteins in micelles and
nanodiscs are solution NMR samples. We will discuss these separately.
9.3.1 Membrane Protein Structure and Function by ssNMR
ssNMR was used initially to study membrane proteins. Some of the lipids have
tendency of aligning with the magnetic field and when they are present in the form
of bicelles they align the proteins inserted inside it. The sample starts behaving like
liquid crystals and it is possible to record experiments on the protein inserted which
are
15 N or
13 C labeled. The preparation of bicelles has active area of the research has
been to replace the detergent from bicelles sample to prepared macro discs which
can align similar to bicelles samples [80–82].
Another area of ssNMR dealing with membrane proteins uses spinning the sample
at magic angle (MAS, spinning sample at 54.7°) to average out dipolar coupling
and reduce chemical shift anisotropy. Membrane proteins embedded in liposomes
(Fig. 27b) are the samples MAS experiments mostly [74]. Either alone or in combination of other NMR non NMR methods, ssNMR at MAS has been used to study
M. Rashid et al.
diphosphate ribose) which are involved in bio-calcification [72]. This area of research
is still open with many more possibilities.
9.3 Membrane Proteins
NMR spectroscopy has been utilized to study proteins, specially where crystallization is difficult such as membrane proteins, intrinsically disordered proteins and
proteins forming fibrils [73–77]. Both solution NMR and ssNMR have been used
to study membrane protein structure, functions and interactions. While purification
membrane proteins are extracted out of the membrane using detergent or similar
molecules, refolded (specially in case of beta barrels proteins) and then reconstituted
in suitable kind of membrane environment for further studies. NMR spectroscopy has
the benefit that it can give both structural and dynamical information of the protein
in native state [74]. The membrane models have evolved with time to make the environment closest to native state. Figure 27 shows some common types of membrane
models which have been used and still being used to prepare membrane proteins,
here adhesion invasion locus (AIL) an outer membrane protein from Yersinia pestis
has been shown inserted inside these membrane models. General membrane models
include, Fig. 27 (a) detergent micelles formed by detergent molecules around protein
where the hydrophobic tail is inside and the headgroup is exposed outside (b) liposomes, which is lipid bilayer without detergent (d and e) nanodiscs are lipid discs
whose sized is controlled by membrane scaffold protein wrapped around the disc
as well as bicelles where the size of the size is controlled by detergent molecules
forming the outer rim of the discs. The structure of the protein taken for example
has been calculated in micelles and nanodiscs [78, 79]. Samples reconstituted in
liposomes, bicelles are studied by ssNMR, while membrane proteins in micelles and
nanodiscs are solution NMR samples. We will discuss these separately.
9.3.1 Membrane Protein Structure and Function by ssNMR
ssNMR was used initially to study membrane proteins. Some of the lipids have
tendency of aligning with the magnetic field and when they are present in the form
of bicelles they align the proteins inserted inside it. The sample starts behaving like
liquid crystals and it is possible to record experiments on the protein inserted which
are
15 N or
13 C labeled. The preparation of bicelles has active area of the research has
been to replace the detergent from bicelles sample to prepared macro discs which
can align similar to bicelles samples [80–82].
Another area of ssNMR dealing with membrane proteins uses spinning the sample
at magic angle (MAS, spinning sample at 54.7°) to average out dipolar coupling
and reduce chemical shift anisotropy. Membrane proteins embedded in liposomes
(Fig. 27b) are the samples MAS experiments mostly [74]. Either alone or in combination of other NMR non NMR methods, ssNMR at MAS has been used to study