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mCherry is one of the most successfully applied monomeric red fluorescent proteins (RFPs) for in vivo and in vitro imaging. However, questions pertaining to the photostability of the RFPs remain and rational further engineering of their photostability requires information about the fluorescence quenching mechanism in solution. To this end, NMR spectroscopic investigations might be helpful, and we present the near-complete backbone NMR chemical shift assignment to aid in this pursuit. [ABSTRACT FROM AUTHOR]

Nuclear spin relaxation is a powerful method for studying molecular dynamics at atomic resolution. Recent methods development in biomolecular NMR spectroscopy has enabled detailed investigations of molecular dynamics that are critical for biological function, with prominent examples addressing allostery, enzyme catalysis, and protein folding. Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used [sup.13]C[alpha](i - 1)/[sup.13]C[alpha](i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i - 1 and i, i and i + 1, etc,), we identified correlated motions that extend through continuous segments of the sequence. We detected significant correlated conformational transitions in the native state of the E140Q mutant of the calmodulin C-terminal domain. Previous work has shown that this domain exchanges between two major conformational states that resemble the functionally relevant open and closed states of the WT protein, with a mean correlation time of [approximately equal to] 20 [micro]s. The present results reveal that an entire u-helix undergoes partial unraveling in a transient and cooperative manner. conformational exchange | NMR | nuclear spin relaxation

The CPMG-based relaxation dispersion techniques used to analyze the conformational exchange processes in the folded and unfolded [F(sub exch)/U(sub exch)] system of the drkN SH3 domain is described. The results show that relaxation dispersion experiments give an extremely sensitive tool to probe conformational exchange processes in unfolded states and to get information on the free energy landscape of such systems.

The measurement of mu-s-ms time scale dynamics of Asn and Gln side chains in proteins is discussed. Many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein were shown to be influenced by a chemical exchange process that could be significant in allowing the rapid binding of hydrophobic ligands to the cavity.

Differences in dynamics seen in retinoid X receptor (RXR) free in solution and in complex with a 14-base-pair DNA half-site have been investigated. Lipari-Szabo analysis was used with an axially symmetric tumbling model to gather information about the exchange contribution to the transverse relaxation rates. When DNA-binding had taken place the order parameters were slightly greater. Dynamics in the millisecond to microsecond range were seen more. The DNA-binding-induced folding transition takes place far from the DNA in an area involved in protein-protein interaction. Thus it may be that it is related to the cooperation of dimeric DNA-binding.

The 9-cis retinoic acid receptor (RXR) and the all-trans retinoic acid receptor (RAR) are members of a family of ligand inducible transcription factors. Nuclear receptors belonging to the Class II sub-family, RXR and RAR both bind to DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The DNA-binding domains of RXR and RAR have zinc-coordinating loop regions that exhibit millisecond to microsecond time scale dynamics. A functional role for the dynamics is suggested by the close association between regions of conformational exchange and dimer interfaces.

A study was conducted to characterize the structure of a photosensory protein from Ectothiorhodopsin halophila called photoactive yellow protein. Multidimensional nuclear magnetic resonance spectroscopy was carried out to determine selected structures and a root-mean-square deviation. Results indicated differences in the crystal structure located in the n-terminus which established lariat-like loop structure.

Several studies are conducted to explain the sequence-specific random coil chemical shits exhibited by the different intrinsically disordered proteins (IDPs). The accuracy of such predictions is shown to be much higher, as compared to that exhibited by the libraries for small peptides of folded proteins.

A method is described for analyzing microsecond to millisecond time scale dynamics of methylgroups in proteins, which have exploited their high abundance and favorable relaxation properties. The results have shown that a cost-effective and quantitative study of protein methyl group dynamics by [super 1]H Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone [super 15]N relaxation dispersion NMR.

An inflection seen in atomic mean square displacements measured as a function of temperature on high resolution spectrometers is described by the onset of methyl group rotations. Molecular dynamics simulations have shown that the apparent transition is because of methyl group rotation entering the infinite instrumental resolution of the spectrometer.

The article explains a simple approach, which is used for the extraction of the Leu side-chain conformations taking place in the excited protein states. The methodology is shown to be extremely useful for the characterization of the structural, as well as the dynamic properties of the different states.

A series of [super 13]C relaxation experiments are used for studying the dynamics of carbonyl and carboxyl groups in protein side chains. The results have provided information on processes on both fast and slower time scales that are relevant for understanding the biological function of these groups.

Triple-resonance NMR methods are used for the direct determination of lysine and arginine side-chain protonation states in [supre 13]C/[super 15]N-labeled proteins. Ideal two-state titration behavior of all lysines has indicated the absence of significant direct charge interactions between the basic residues.

[super 13]C NMR chemical shift data is used for individual aspartate and glutamate side chain carboxylate groups to accurately detect site-specific protonation equilibria in a variant of the B1 domain of protein G (PGB1-QDD). The selective study of charge-charge interactions and the comparison of the proton binding affinity (p[K.sub.a]) values at high and low salt have described the importance of Coulombic interactions in shifting p[K.sub.a] values.

A study was conducted, using heteronuclear NMR spectroscopy, to characterize L12 conformation and dynamics in solution and on the ribosome. The result shows no persistent contacts between the two C-terminal domains (CTDs), or between the N-terminal domains (NTDs) and CTD in the L2 dimer in solution.

The application of (super 13)CH3 transverse relaxation dispersion NMR experiments to the study of confirmational exchange in a cavity mutant form of T4 lysozyme labeled with 13C at methyl positions using (super 13)CH3- pyruvate is described. The findings demonstrate the general applicability of methyl relaxation dispersion measurements for the investigation of millisecond time scale protein motions at large number of side-chain positions.

Relaxation dispersion NMR measurements were used to probe slow time scale dynamics at methyl-containing side chains in proteins.