Editing Protein quaternary structure (section)

==Structure Determination==

Protein quaternary structure can be determined using a variety of experimental techniques that require a sample of protein in a variety of experimental conditions. The experiments often provide an estimate of the mass of the native protein and, together with knowledge of the masses and/or stoichiometry of the subunits, allow the quaternary structure to be predicted with a given accuracy. It is not always possible to obtain a precise determination of the subunit composition for a variety of reasons.

The number of subunits in a protein complex can often be determined by measuring the hydrodynamic molecular volume or mass of the intact complex, which requires native solution conditions.  For ''folded'' proteins, the mass can be inferred from its volume using the partial specific volume of 0.73 ml/g.  However, volume measurements are less certain than mass measurements, since ''unfolded'' proteins appear to have a much larger volume than folded proteins; additional experiments are required to determine whether a protein is unfolded or has formed an oligomer.

=== Common techniques used to study protein quaternary structure ===

* Ultracentrifugation
* Surface-induced dissociation mass spectrometry<ref name = "Stiving_2019">{{cite journal | vauthors = Stiving AQ, VanAernum ZL, Busch F, Harvey SR, Sarni SH, Wysocki VH | title = Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure | journal = Analytical Chemistry | volume = 91 | issue = 1 | pages = 190–209 | date = January 2019 | pmid = 30412666 | pmc = 6571034 | doi = 10.1021/acs.analchem.8b05071 | department = review }}</ref>
* Coimmunoprecipation<ref name = "Milligan_2005">{{cite journal | vauthors = Milligan G, Bouvier M | title = Methods to monitor the quaternary structure of G protein-coupled receptors | journal = The FEBS Journal | volume = 272 | issue = 12 | pages = 2914–2925 | date = June 2005 | pmid = 15955052 | doi = 10.1111/j.1742-4658.2005.04731.x | s2cid = 23274563 | department = review }}</ref>
* [[Förster resonance energy transfer|FRET]]<ref name = "Milligan_2005" /><ref name = "Raicu_2013">{{cite journal | vauthors = Raicu V, Singh DR | title = FRET spectrometry: a new tool for the determination of protein quaternary structure in living cells | journal = Biophysical Journal | volume = 105 | issue = 9 | pages = 1937–1945 | date = November 2013 | pmid = 24209838 | pmc = 3824708 | doi = 10.1016/j.bpj.2013.09.015 | bibcode = 2013BpJ...105.1937R | department = primary }}</ref>
* Nuclear Magnetic Resonance (NMR)<ref name="Prischi_2016">{{cite book | vauthors = Prischi F, Pastore A | title = Advanced Technologies for Protein Complex Production and Characterization | chapter = Application of Nuclear Magnetic Resonance and Hybrid Methods to Structure Determination of Complex Systems | series = Advances in Experimental Medicine and Biology | volume = 896 | pages = 351–368 | date = 2016 | pmid = 27165336 | doi = 10.1007/978-3-319-27216-0_22 | isbn = 978-3-319-27214-6 | department = review }}</ref><ref name="Wells_2018">{{cite book | vauthors = Wells JN, Marsh JA | title = Protein Complex Assembly | chapter = Experimental Characterization of Protein Complex Structure, Dynamics, and Assembly | series = Methods in Molecular Biology | volume = 1764 | pages = 3–27 | date = 2018 | pmid = 29605905 | doi = 10.1007/978-1-4939-7759-8_1 | isbn = 978-1-4939-7758-1 | quote = Section 4: Nuclear Magnetic Resonance Spectroscopy | department = review }}</ref>

===Direct mass measurement of intact complexes===

* Sedimentation-equilibrium [[analytical ultracentrifugation]] 
* [[electrospray ionization|Electrospray]] [[mass spectrometry]]
* [[Mass Spectrometric Immunoassay]] MSIA

===Direct size measurement of intact complexes===

* [[Rayleigh scattering|Static light scattering]]
* [[Size exclusion chromatography]] (requires calibration)
* [[Dual polarisation interferometry]]

===Indirect size measurement of intact complexes===

* Sedimentation-velocity [[analytical ultracentrifugation]] (measures the translational [[diffusion constant]])
* [[Dynamic light scattering]] (measures the translational [[diffusion constant]])
* Pulsed-gradient [[protein nuclear magnetic resonance]] (measures the translational [[diffusion constant]])
* [[Fluorescence polarization]] (measures the rotational [[diffusion constant]])
* [[Dielectric relaxation]] (measures the rotational [[diffusion constant]])
* [[Dual polarisation interferometry]] (measures the size and the density of the complex)

Methods that measure the mass or volume under [[Denaturation (biochemistry)|unfolding]] conditions (such as 
[[Matrix-assisted laser desorption/ionization|MALDI-TOF]] [[mass spectrometry]] and [[Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis|SDS-PAGE]]) are generally not useful, since non-native conditions usually cause the complex to dissociate into monomers. However, these may sometimes be applicable; for example, the experimenter may apply SDS-PAGE after first treating the intact complex with chemical [[cross-link]] reagents.