Editing Protein folding (section)

== Experimental techniques for studying protein folding ==
While inferences about protein folding can be made through [[Phi value analysis|mutation studies]], typically, experimental techniques for studying protein folding rely on the [[Equilibrium unfolding|gradual unfolding]] or folding of proteins and observing conformational changes using standard non-crystallographic techniques.

===X-ray crystallography===
[[File:X ray diffraction.png|thumb|Steps of [[X-ray crystallography]]|321x321px]]
[[X-ray crystallography]] is one of the more efficient and important methods for attempting to decipher the three dimensional configuration of a folded protein.<ref name="Cowtan_2001">{{cite encyclopedia | url = http://people.bu.edu/mfk/restricted566/phaseproblem.pdf | title = Phase Problem in X-ray Crystallography, and Its Solution | last = Cowtan | first = Kevin | name-list-style = vanc | date = 2001 | encyclopedia = Encyclopedia of Life Sciences |publisher=Macmillan Publishers Ltd, Nature Publishing Group|access-date=November 3, 2016}}</ref> To be able to conduct X-ray crystallography, the protein under investigation must be located inside a crystal lattice. To place a protein inside a crystal lattice, one must have a suitable solvent for crystallization, obtain a pure protein at supersaturated levels in solution, and precipitate the crystals in solution.<ref>{{cite book | url=https://books.google.com/books?id=Jobr7svN0IIC&pg=PR5 |title = Principles of Protein X-Ray Crystallography | last = Drenth | first = Jan | name-list-style = vanc | date = 2007-04-05 | publisher = Springer Science & Business Media | isbn = 978-0-387-33746-3 }}</ref> Once a protein is crystallized, X-ray beams can be concentrated through the crystal lattice which would diffract the beams or shoot them outwards in various directions. These exiting beams are correlated to the specific three-dimensional configuration of the protein enclosed within. The X-rays specifically interact with the electron clouds surrounding the individual atoms within the protein crystal lattice and produce a discernible diffraction pattern.<ref name="Fersht_1999">{{cite book |url=https://books.google.com/books?id=QdpZz_ahA5UC&pg=PR20 |title=Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding | last = Fersht | first = Alan | name-list-style = vanc | date = 1999 | publisher = Macmillan | isbn = 978-0-7167-3268-6 }}</ref> Only by relating the electron density clouds with the amplitude of the X-rays can this pattern be read and lead to assumptions of the phases or phase angles involved that complicate this method.<ref>{{cite journal |doi=10.1107/S0907444903017815 |title=The phase problem |journal=Acta Crystallographica Section D |volume=59 |issue=11 |pages=1881–90 |year=2003 |last1=Taylor |first1=Garry | name-list-style = vanc |pmid=14573942 |doi-access=free |bibcode=2003AcCrD..59.1881T }}</ref> Without the relation established through a mathematical basis known as [[Fourier transform]], the "[[phase problem]]" would render predicting the diffraction patterns very difficult.<ref name="Fersht_1999" /> Emerging methods like [[multiple isomorphous replacement]] use the presence of a heavy metal ion to diffract the X-rays into a more predictable manner, reducing the number of variables involved and resolving the phase problem.<ref name="Cowtan_2001" />

===Fluorescence spectroscopy===
[[Fluorescence spectroscopy]] is a highly sensitive method for studying the folding state of proteins. Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their [[quantum yield]]s are high enough to give good fluorescence signals. Both Trp and Tyr are excited by a wavelength of 280&nbsp;nm, whereas only Trp is excited by a wavelength of 295&nbsp;nm. Because of their aromatic character, Trp and Tyr residues are often found fully or partially buried in the hydrophobic core of proteins, at the interface between two protein domains, or at the interface between subunits of oligomeric proteins. In this apolar environment, they have high quantum yields and therefore high fluorescence intensities. Upon disruption of the protein's tertiary or quaternary structure, these side chains become more exposed to the hydrophilic environment of the solvent, and their quantum yields decrease, leading to low fluorescence intensities. For Trp residues, the wavelength of their maximal fluorescence emission also depend on their environment.

Fluorescence spectroscopy can be used to characterize the [[equilibrium unfolding]] of proteins by measuring the variation in the intensity of fluorescence emission or in the wavelength of maximal emission as functions of a denaturant value.<ref name="pmid=26607240">{{cite journal | vauthors = Bedouelle H | title = Principles and equations for measuring and interpreting protein stability: From monomer to tetramer | journal = Biochimie | volume = 121 | pages = 29–37 | date = February 2016 | pmid = 26607240 | doi = 10.1016/j.biochi.2015.11.013 }}</ref><ref>{{cite journal | vauthors = Monsellier E, Bedouelle H | title = Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength | journal = Protein Engineering, Design & Selection | volume = 18 | issue = 9 | pages = 445–56 | date = September 2005 | pmid = 16087653 | doi = 10.1093/protein/gzi046 | doi-access = free }}</ref> The denaturant can be a chemical molecule (urea, guanidinium hydrochloride), temperature, pH, pressure, etc. The equilibrium between the different but discrete protein states, i.e. native state, intermediate states, unfolded state, depends on the denaturant value; therefore, the global fluorescence signal of their equilibrium mixture also depends on this value. One thus obtains a profile relating the global protein signal to the denaturant value. The profile of equilibrium unfolding may enable one to detect and identify intermediates of unfolding.<ref>{{cite journal | vauthors = Park YC, Bedouelle H | title = Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions | journal = The Journal of Biological Chemistry | volume = 273 | issue = 29 | pages = 18052–9 | date = July 1998 | pmid = 9660761 | doi = 10.1074/jbc.273.29.18052 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Ould-Abeih MB, Petit-Topin I, Zidane N, Baron B, Bedouelle H | title = Multiple folding states and disorder of ribosomal protein SA, a membrane receptor for laminin, anticarcinogens, and pathogens | journal = Biochemistry | volume = 51 | issue = 24 | pages = 4807–21 | date = June 2012 | pmid = 22640394 | doi = 10.1021/bi300335r }}</ref> General equations have been developed by Hugues Bedouelle to obtain the thermodynamic parameters that characterize the unfolding equilibria for homomeric or heteromeric proteins, up to trimers and potentially tetramers, from such profiles.<ref name="pmid=26607240"/> Fluorescence spectroscopy can be combined with fast-mixing devices such as [[stopped flow]], to measure protein folding kinetics,<ref>{{cite journal | vauthors = Royer CA | title = Probing protein folding and conformational transitions with fluorescence | journal = Chemical Reviews | volume = 106 | issue = 5 | pages = 1769–84 | date = May 2006 | pmid = 16683754 | doi = 10.1021/cr0404390 }}</ref> generate a [[chevron plot]] and derive a [[Phi value analysis]].

===Circular dichroism===
{{Main|Circular dichroism}}

[[Circular dichroism]] is one of the most general and basic tools to study protein folding. Circular dichroism spectroscopy measures the absorption of [[circular polarization|circularly polarized light]]. In proteins, structures such as [[alpha helix|alpha helices]] and [[beta sheets]] are chiral, and thus absorb such light. The absorption of this light acts as a marker of the degree of foldedness of the protein ensemble. This technique has been used to measure [[equilibrium unfolding]] of the protein by measuring the change in this absorption as a function of denaturant concentration or [[temperature]]. A denaturant melt measures the [[Thermodynamic free energy|free energy]] of unfolding as well as the protein's m value, or denaturant dependence. A temperature melt measures the [[Denaturation midpoint|denaturation temperature]] (Tm) of the protein.<ref name="pmid=26607240" /> As for fluorescence spectroscopy, circular-dichroism spectroscopy can be combined with fast-mixing devices such as [[stopped flow]] to measure protein folding [[Chemical kinetics|kinetics]] and to generate [[chevron plot]]s.

===Vibrational circular dichroism of proteins===
The more recent developments of [[vibrational circular dichroism]] (VCD) techniques for proteins, currently involving [[Fourier transform]] (FT) instruments, provide powerful means for determining protein conformations in solution even for very large protein molecules. Such VCD studies of proteins can be combined with [[X-ray diffraction]] data for protein crystals, [[FT-IR]] data for protein solutions in heavy water (D<sub>2</sub>O), or [[Quantum chemistry|quantum computations]].

===Protein nuclear magnetic resonance spectroscopy===
{{Main|Protein NMR}}

Protein [[nuclear magnetic resonance]] (NMR) is able to collect protein structural data by inducing a magnet field through samples of concentrated protein. In NMR, depending on the chemical environment, certain nuclei will absorb specific radio-frequencies.<ref name=":1">{{cite journal | vauthors = Wüthrich K | title = Protein structure determination in solution by NMR spectroscopy | journal = The Journal of Biological Chemistry | volume = 265 | issue = 36 | pages = 22059–62 | date = December 1990 | doi = 10.1016/S0021-9258(18)45665-7 | pmid = 2266107 | url = http://www.jbc.org/content/265/36/22059 | doi-access = free }}</ref><ref name=":0">{{cite journal | vauthors = Zhuravleva A, Korzhnev DM | title = Protein folding by NMR | journal = Progress in Nuclear Magnetic Resonance Spectroscopy | volume = 100 | pages = 52–77 | date = May 2017 | pmid = 28552172 | doi = 10.1016/j.pnmrs.2016.10.002 | bibcode = 2017PNMRS.100...52Z | url = http://www.sciencedirect.com/science/article/pii/S0079656516300280 }}</ref> Because protein structural changes operate on a time scale from ns to ms, NMR is especially equipped to study intermediate structures in timescales of ps to s.<ref name=":5">{{cite book |doi=10.1016/b978-0-12-411636-8.00006-7 |chapter=Protein Functional Dynamics in Multiple Timescales as Studied by NMR Spectroscopy |title=Dynamics of Proteins and Nucleic Acids |series=Advances in Protein Chemistry and Structural Biology |year=2013 |last1=Ortega |first1=Gabriel |last2=Pons |first2=Miquel |last3=Millet |first3=Oscar |volume=92 |pages=219–251 |pmid=23954103 |isbn=9780124116368 }}</ref> Some of the main techniques for studying proteins structure and non-folding protein structural changes include [[Two-dimensional nuclear magnetic resonance spectroscopy|COSY]], [[Two-dimensional nuclear magnetic resonance spectroscopy|TOCSY]], [[Heteronuclear single quantum coherence spectroscopy|HSQC]], [[Relaxation (NMR)|time relaxation]] (T1 & T2), and [[Nuclear Overhauser effect|NOE]].<ref name=":1" /> NOE is especially useful because magnetization transfers can be observed between spatially proximal hydrogens are observed.<ref name=":1" /> Different NMR experiments have varying degrees of timescale sensitivity that are appropriate for different protein structural changes. NOE can pick up bond vibrations or side chain rotations, however, NOE is too sensitive to pick up protein folding because it occurs at larger timescale.<ref name=":5" />[[File:Protein Structural changes timescale matched with NMR experiments.png|thumb|350x350px|Timescale of protein structural changes matched with NMR experiments. For protein folding, CPMG Relaxation Dispersion (CPMG RD) and chemical exchange saturation transfer (CEST) collect data in the appropriate timescale.]]

Because protein folding takes place in about 50 to 3000 s<sup>−1</sup> CPMG Relaxation dispersion and [[Magnetization transfer|chemical exchange saturation transfer]] have become some of the primary techniques for NMR analysis of folding.<ref name=":0" /> In addition, both techniques are used to uncover excited intermediate states in the protein folding landscape.<ref name=":4">{{cite journal | vauthors = Vallurupalli P, Bouvignies G, Kay LE | title = Studying "invisible" excited protein states in slow exchange with a major state conformation | journal = Journal of the American Chemical Society | volume = 134 | issue = 19 | pages = 8148–61 | date = May 2012 | pmid = 22554188 | doi = 10.1021/ja3001419 }}</ref> To do this, CPMG Relaxation dispersion takes advantage of the [[spin echo]] phenomenon. This technique exposes the target nuclei to a 90 pulse followed by one or more 180 pulses.<ref name=":2">{{cite journal | vauthors = Neudecker P, Lundström P, Kay LE | title = Relaxation dispersion NMR spectroscopy as a tool for detailed studies of protein folding | journal = Biophysical Journal | volume = 96 | issue = 6 | pages = 2045–54 | date = March 2009 | pmid = 19289032 | pmc = 2717354 | doi = 10.1016/j.bpj.2008.12.3907 | bibcode = 2009BpJ....96.2045N }}</ref> As the nuclei refocus, a broad distribution indicates the target nuclei is involved in an intermediate excited state. By looking at Relaxation dispersion plots the data collect information on the thermodynamics and kinetics between the excited and ground.<ref name=":2" /><ref name=":4" /> Saturation Transfer measures changes in signal from the ground state as excited states become perturbed. It uses weak radio frequency irradiation to saturate the excited state of a particular nuclei which transfers its saturation to the ground state.<ref name=":0" /> This signal is amplified by decreasing the magnetization (and the signal) of the ground state.<ref name=":0" /><ref name=":4" />

The main limitations in NMR is that its resolution decreases with proteins that are larger than 25 kDa and is not as detailed as [[X-ray crystallography]].<ref name=":0" /> Additionally, protein NMR analysis is quite difficult and can propose multiple solutions from the same NMR spectrum.<ref name=":1" />

In a study focused on the folding of an [[amyotrophic lateral sclerosis]] involved protein [[SOD1]], excited intermediates were studied with relaxation dispersion and Saturation transfer.<ref name=":3">{{cite journal | vauthors = Sekhar A, Rumfeldt JA, Broom HR, Doyle CM, Sobering RE, Meiering EM, Kay LE | title = Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 45 | pages = E6939–E6945 | date = November 2016 | pmid = 27791136 | pmc = 5111666 | doi = 10.1073/pnas.1611418113 | bibcode = 2016PNAS..113E6939S | doi-access = free }}</ref> SOD1 had been previously tied to many disease causing mutants which were assumed to be involved in protein aggregation, however the mechanism was still unknown. By using Relaxation Dispersion and Saturation Transfer experiments many excited intermediate states were uncovered misfolding in the SOD1 mutants.<ref name=":3" />

=== Dual-polarization interferometry ===
{{Main|Dual-polarization interferometry}}
[[Dual polarisation interferometry]] is a surface-based technique for measuring the optical properties of molecular layers. When used to characterize protein folding, it measures the [[protein conformation|conformation]] by determining the overall size of a monolayer of the protein and its density in real time at sub-Angstrom resolution,<ref name="CrossFreeman2008">{{cite book |doi=10.1002/9780470061565.hbb055 |chapter=Dual Polarization Interferometry: A Real-Time Optical Technique for Measuring (Bio)molecular Orientation, Structure and Function at the Solid/Liquid Interface |title=Handbook of Biosensors and Biochips |year=2008 |last1=Cross |first1=Graham H. |last2=Freeman |first2=Neville J. |last3=Swann |first3=Marcus J. | name-list-style = vanc |isbn=978-0-470-01905-4 }}</ref> although real-time measurement of the kinetics of protein folding are limited to processes that occur slower than ~10&nbsp;Hz. Similar to [[circular dichroism]], the stimulus for folding can be a denaturant or [[temperature]].

=== Studies of folding with high time resolution ===
The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. Experimenters rapidly trigger the folding of a sample of unfolded protein and observe the resulting [[protein dynamics|dynamics]]. Fast techniques in use include [[neutron scattering]],<ref name="Callaway">{{cite journal | vauthors = Bu Z, Cook J, Callaway DJ | title = Dynamic regimes and correlated structural dynamics in native and denatured alpha-lactalbumin | journal = Journal of Molecular Biology | volume = 312 | issue = 4 | pages = 865–73 | date = September 2001 | pmid = 11575938 | doi = 10.1006/jmbi.2001.5006 }}</ref> ultrafast mixing of solutions, photochemical methods, and [[Temperature jump|laser temperature jump spectroscopy]]. Among the many scientists who have contributed to the development of these techniques are Jeremy Cook, Heinrich Roder, Terry Oas, [[Harry Gray (chemist)|Harry Gray]], [[Martin Gruebele]], Brian Dyer, William Eaton, [[Sheena Radford]], [[Chris Dobson]], [[Alan Fersht]], [[Bengt Nölting]] and Lars Konermann.

=== Proteolysis ===
[[Proteolysis]] is routinely used to probe the fraction unfolded under a wide range of solution conditions (e.g. [[fast parallel proteolysis (FASTpp)]].<ref name="Minde">{{cite journal | vauthors = Minde DP, Maurice MM, Rüdiger SG | title = Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp | journal = PLOS ONE | volume = 7 | issue = 10 | pages = e46147 | year = 2012 | pmid = 23056252 | pmc = 3463568 | doi = 10.1371/journal.pone.0046147 | bibcode = 2012PLoSO...746147M | doi-access = free }}</ref><ref name="Park">{{cite journal | vauthors = Park C, Marqusee S | title = Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding | journal = Nature Methods | volume = 2 | issue = 3 | pages = 207–12 | date = March 2005 | pmid = 15782190 | doi = 10.1038/nmeth740 | s2cid = 21364478 }}</ref>

=== Single-molecule force spectroscopy ===
Single molecule techniques such as optical tweezers and AFM have been used to understand protein folding mechanisms of isolated proteins as well as proteins with chaperones.<ref name="pmid24001118">{{cite journal | vauthors = Mashaghi A, Kramer G, Lamb DC, Mayer MP, Tans SJ | title = Chaperone action at the single-molecule level | journal = Chemical Reviews | volume = 114 | issue = 1 | pages = 660–76 | date = January 2014 | pmid = 24001118 | doi = 10.1021/cr400326k }}</ref> [[Optical tweezers]] have been used to stretch single protein molecules from their C- and N-termini and unfold them to allow study of the subsequent refolding.<ref>{{cite journal | vauthors = Jagannathan B, Marqusee S | title = Protein folding and unfolding under force | journal = Biopolymers | volume = 99 | issue = 11 | pages = 860–9 | date = November 2013 | pmid = 23784721 | pmc = 4065244 | doi = 10.1002/bip.22321 }}</ref> The technique allows one to measure folding rates at single-molecule level; for example, optical tweezers have been recently applied to study folding and unfolding of proteins involved in blood coagulation. [[von Willebrand factor]] (vWF) is a protein with an essential role in blood clot formation process. It discovered – using single molecule optical tweezers measurement – that calcium-bound vWF acts as a shear force sensor in the blood. Shear force leads to unfolding of the A2 domain of vWF, whose refolding rate is dramatically enhanced in the presence of calcium.<ref>{{cite journal | vauthors = Jakobi AJ, Mashaghi A, Tans SJ, Huizinga EG | title = Calcium modulates force sensing by the von Willebrand factor A2 domain | journal = Nature Communications | volume = 2 | pages = 385 | date = July 2011 | pmid = 21750539 | pmc = 3144584 | doi = 10.1038/ncomms1385 | bibcode = 2011NatCo...2..385J }}</ref> Recently, it was also shown that the simple src [[SH3 domain]] accesses multiple unfolding pathways under force.<ref>{{cite journal | vauthors = Jagannathan B, Elms PJ, Bustamante C, Marqusee S | title = Direct observation of a force-induced switch in the anisotropic mechanical unfolding pathway of a protein | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 44 | pages = 17820–5 | date = October 2012 | pmid = 22949695 | pmc = 3497811 | doi = 10.1073/pnas.1201800109 | bibcode = 2012PNAS..10917820J | doi-access = free }}</ref>

=== Biotin painting ===
Biotin painting enables condition-specific cellular snapshots of (un)folded proteins. Biotin 'painting' shows a bias towards predicted [[Intrinsically disordered proteins]].<ref name="Biotinylation by proximity labelling favours unfolded proteins">{{cite journal | vauthors = Minde DP, Ramakrishna M, Lilley KS | title = Biotinylation by proximity labelling favours unfolded proteins | journal = bioRxiv | year = 2018 | doi = 10.1101/274761 | doi-access = free | url = https://www.biorxiv.org/content/biorxiv/early/2018/09/13/274761.full.pdf }}</ref>