<P> X-ray crystallography is one of the more efficient and important methods for attempting to decipher the three dimensional configuration of a folded protein . 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 . 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 . 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 . Without the relation established through a mathematical basis known as Fourier transform, the "phase problem" would render predicting the diffraction patterns very difficult . 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 . </P> <P> 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 yields are high enough to give good fluorescence signals . Both Trp and Tyr are excited by a wavelength of 280 nm, whereas only Trp is excited by a wavelength of 295 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 . </P> <P> 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 . 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 . 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 . Fluorescence spectroscopy can be combined with fast - mixing devices such as stopped flow, to measure protein folding kinetics, generate a chevron plot and derive a Phi value analysis . </P> <P> Circular dichroism is one of the most general and basic tools to study protein folding . Circular dichroism spectroscopy measures the absorption of circularly polarized light . In proteins, structures such as 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 free energy of unfolding as well as the protein's m value, or denaturant dependence . A temperature melt measures the melting temperature (T) of the protein . As for fluorescence spectroscopy, circular - dichroism spectroscopy can be combined with fast - mixing devices such as stopped flow to measure protein folding kinetics and to generate chevron plots . </P>

Why do proteins need to be so complex