Structural/functional types Functional classes Functional subclasses Detection methods

List of all detection methods

Detection methodDescription
Analytical ultracentrifugation This method is based on the visualization the behavior of a particle in real time during sedimentation, allowing extremely accurate determination of hydrodynamic and thermodynamic parameters, as well as estimation of the molecular weight of proteins. These can then be compared with the size of a globular protein of the same mass. It is also useful in monitoring changes in conformation of the proteins.
Atomic force microscopy (AFM) High-speed atomic force microscopy (AFM) is a type of scanning probe microscopy that allows direct visualization of biological samples in physiological conditions at nanometer resolution. Slow AFM produces about 1 frame per minute whereas high-speed AFM produces about 10 frames per second. Due to its high imaging rate, high-speed AFM can capture the fluctuations of intrinsically disordered proteins.
Circular dichroism (CD) spectroscopy, far-UV Detects the presence or absence of ordered secondary structure and can be used to estimate the secondary structure content in a given protein. CD spectra of natively unfolded proteins, being measured in the far ultraviolet region, show a large negative peak around 200 nm, as well as a value near zero at 220 nm in partially or fully unstructured proteins. These values are distinct enough to distinguish them from ordered proteins.
Circular dichroism (CD) spectroscopy, near-UV Indications of protein tertiary structure can be obtained by assaying the solvent accessibility of one type of optically active chromophores in proteins (side groups of aromatic amino acid residues. CD spectra in the near ultraviolet region (250 - 350 nm), also called the aromatic region, reflect the symmetry of the environment of aromatic amino acid residues. A highly symmetrical environment indicates solvent accessibility. Protein denaturation (where the normally protected hydrophobic core of a globular protein becomes exposed to the solvent) can be monitored by changes in the near-UV CD spectrum.
Dynamic light scattering Passes a beam of light through a sample, and then measures the extent of the scattering. This provides information on the hydrodynamic radius of the protein, which is dependent upon the size and conformation of the protein
Electrophoretic Mobility Shift Assay (EMSA)  
ESI-FTICR mass spectrometry  
Fluorescence, intrinsic Three amino acids exhibit intrinsic fluorescence: tryptophan, tyrosine and phenylalanine (ranked here in order of decreasing quantum yield) with tryptophan being the most commonly used. Quenching of tyrosine fluorescence can be due to ionization, to proximity to amide or carboxyl groups, or to the energy transfer to tryptophan. Application of intrinsic fluorescence to the study of protein conformational analysis relies on the fact that the parameters of tryptophan emission (intensity and wavelength of maximal fluorescence) depend essentially on environmental factors, including solvent polarity, pH, and presence or absence of quenchers. For example, a completely solvated tryptophan residue (e.g., free tryptophan in water or tryptophan in an unfolded polypeptide chain) has a maximum fluorescence in the vicinity of 350 nm, but embedding in the nonpolar interior of a compact globular protein results in a characteristic blue shift.
Fluorescence polarization/anisotropy Measurement of the difference between the fluorescent intensities of parallel and perpendicular polarization when compared to the initial, excitation polarization. These measurements can be done for internal chromophores (i.e. tryptophan, tyrosine, phenylalanine) as well as for external chromophores (covalently bound fluorescent labels or dyes). They can give information on speed of chromophore rotation within the molecule and thus provide information on the mobility and orientation of the protein.
Fluorescence resonance energy transfer (FRET) Monitors the irradiative transfer of energy between two chromophores. This is used to determine the distance between the two fluorescence labels. Conformational flexibility (and hence disorder) is observed as changes in this distance.
Fluorescent dynamic quenching The use of chemical or physical means to depopulate the excited state without allowing fluorescence emission. It can provide information on the accessibility of internal chromophores (i.e. tryptophan, tyrosine, phenylalanine) to solvent, and thus describes the relative compactness and dynamics of a protein molecule
Fluorescent probes The rigid structure of a well-folded protein will not allow a fluorescent probe (usually a large hydrophobic molecule) into the folded core, while a molten globular structure will allow the probe to penetrate. Incorporation of the probe into the core alters the environment of the probe, which can be detected as changes in fluorescence.
Fourier transform infrared spectroscopy (FTIR), aka infrared spectroscopy) Presents information on the vibrational movements of functional groups due to bond stretching, change of bond angles, and other types of motion. It can then be compared to experimental data obtained from defined secondary structures (such as alpha helices and beta sheets), and thus can identify secondary characteristics outside the realm of these ordered sets. FTIR differs from standard IR in that an interferometer is used and signal data is processed using a Fourier transform algorithm.
High relative B-factor Refers to the degree in which the electron density is spread out for individual atoms or groups of atoms within a sample. Displacement of atoms results in higher B-factors, which indicate higher relative disorder.
Hydrogen-deuterium exchange Monitors the exchange rate between deuterium in the solvent and hydrogen in the main chain amides. These rates are affected by hydrogen bonding, accessibility of the amides to the surface, and flexibility of the protein. Faster exchange rates indicate the more flexible (and potentially disordered) protein.
Immunochemistry Immunochemical methods may also be applied toward the elucidation of protein disorder. Important to this discussion, the immunoglobulins obtained against a given protein may be specific for different levels of macromolecule: the primary structure, the secondary structure, or the tertiary structure. Thus, antibodies may be successfully used to study the structural state of a protein.
Mass spectrometry-based high resolution hydrogen-deuterium exchange Measures the hydrogen-deuterium exchange rate for a given protein, but does not require crystals (as in X-ray crystallography) and is not limited to protein size and sample concentration (as in NMR). These exchange rates are affected by hydrogen bonding, accessibility of the protein surface, and flexibility of the protein. A faster exchange rate indicates that the protein is less stable, more flexible and may be disordered.
Nuclear magnetic resonance (NMR) Heteronuclear multidimensional NMR is an extremely powerful technique for three-dimensional structure determination of proteins in solution. Regions of high backbone flexibility have reduced chemical shift dispersion indicative of disorder. Spin relaxation and nuclear Overhauser effect measurements can provide information on the molecular dynamics of the disordered region. Recent advances have allowed the complete assignment of resonances for several unfolded proteins as well as some disordered fragments of some folded proteins.
Optical rotatory dispersion Measures the optical dispersion caused by a protein, in which the wavelength of the light is varied. This takes advantage of the fact that right and left handed molecules cause different rotational effects on the polarized light. This can be used to determine the presence and amount of secondary structures, such as alpha helices.
Radio-labelled Enzyme Activity Assay Detection of enzymatic activity using radio-labelled proteins or substrates. Reaction products are separated on gels by electrophoresis, phosphor-imaged, then visualized and quantified using relevant computer software. Comparing activity of native vs. modified proteins can indicate protein function(s).
Raman optical activity Raman optical activity can be used to provide information on secondary structure. Raman optical activity provides a means to distinguish between dynamic (flexible chain) and static disorder (structured domain tethered by a hinge region).
Raman spectroscopy Raman spectroscopy can be used to measure secondary structure content in the same way that is used for FTIR. The Raman spectrum of an unfolded peptide or protein is distinct from that of other forms of secondary.
Rotory shadowing electron microscopy In this technique, a protein sample is coated with a heavy metal, such as platinum. These metals block the electron beams used in TEM, and can reveal the shape of the protein. As the source of the metal lies at an angle to the sample surface, the thickness of the accumulated metal varies with the surface topography. By rotating the sample while coating with the metal, the 3D characteristics of the sample can be observed in high resolution.
SDS-PAGE gel, Aberrant mobility on Aberrant mobility on SDS-PAGE gels is an indication that the protein under study is disordered. However, this is not a definitive method and should be treated as supporting evidence.
SDS-PAGE/Pull-down Assays Pull-down assays can elucidate protein-protein interactions. A tagged \"bait\" protein captures (\"pulls down\") its \"prey\" binding partner protein. SDS-PAGE visualizes the results of the assay. GST and histidine are commonly used tags.
Sensitivity to proteolysis Unstructured proteins have increased proteolytic degradation rates in vitro due to their increased flexibility which allows unfettered access to the chain by proteases.
Size exclusion/gel filtration chromatography Separates proteins on the basis of their molecular size and hydrodynamic volumes.
Small-angle neutron scattering (SANS) Neutrons are passed through an aqueous solution containing a protein sample, which causes a scattering effect. The results can then be used to obtain details on the size, shape, and orientation of the protein. Neutron scattering provides a means of measuring on scales much smaller than light scattering because of the smaller size of the neutron wavelengths.
Small-angle X-ray scattering (SAXS) The degree of globularity, which reflects the presence or absence of a tightly packed core in a protein molecule, can be extracted from the analysis of SAXS data in the form of a Kratky plot, whose shape is sensitive to the conformational state of the scattering protein molecules. It has been shown that a scattering curve in the Kratky coordinates has a characteristic maximum for globular proteins in either their native or molten globule states (i.e., states with globular structure). However, if a protein is completely unfolded or in a premolten globule conformation (i.e., with no globular structure), such a maximum will be absent.
Stability at pH extremes Intrinsically unstructured proteins, typically with extended forms and often with high percentages of charged residues, do not undergo large-scale structural changes when subjected to changes in pH. Unlike intrinsically unfolded proteins, structured proteins typically contain large numbers of hydrophobic residues that upon exposure due to pH-induced loosening of the structure, promote aggregation and precipitation.
Stability at thermal extremes Structured proteins are generally denatured by heat, whereas high temperatures might induce folding in disordered proteins. This induced folding is believed to be a result of increased hydrophobic interactions, which leads to a greater hydrophobic driving force for protein folding.
Static light scattering This method is based on passing a beam of light through a sample, and then measuring the extent of the scattering at one specific scattering angle. This provides information on the hydrodynamic radius of the protein, which is dependent upon the size and conformation of the protein.
Synchrotron radiation circular dichroism (SRCD)  
Viscometry The viscosity of a sample, which is the degree to which it resists flow under an applied force, is affected by the molecular weight of a protein and its shape and conformation. By comparing the behavior of a protein to those of known structure and similar size, the relative disorder of a protein can be determined.
X-ray crystallography Missing electron density of backbone atoms found in three-dimensional structures determined by X-ray crystallography can indicate disordered regions. The increased flexibility of atoms in such regions leads to noncoherent X-ray scattering, preventing them from being observed.
zz FTIR (Duplicate, to be deleted) Duplicate method, unable to remove (3/25/10, JPB).
zz IR/FTIR (Duplicate, to be deleted) Duplicate method, unable to remove (3/25/10, JPB).

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