Global protein dynamics as communication sensors in peptide synthetase domains.

Biological activity is governed by the timely redistribution of molecular interactions, and static structural snapshots often appear insufficient to provide the molecular determinants that choreograph communication. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites, where a dynamic domain organization challenges rational design to produce new pharmaceuticals. Using a nuclear magnetic resonance (NMR) atomic-level readout of biochemical transformations, we demonstrate that global structural fluctuations help promote substrate-dependent communication and allosteric responses, and impeding these global dynamics by a point-site mutation hampers allostery and molecular recognition. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions, and they provide new perspectives on mechanisms of allostery, protein communication, and NRPS synthesis.

Minimizing Pervasive Artifacts in 4D Covariance Maps for Protein Side Chain NMR Assignments.

Nuclear magnetic resonance (NMR) is a mainstay of biophysical studies that provides atomic level readouts to formulate molecular mechanisms. Side chains are particularly important to derive mechanisms involving proteins as they carry functional groups, but NMR studies of side chains are often limited by challenges in assigning their signals. Here, we designed a novel computational method that combines spectral derivatives and matrix square-rooting to produce reliable 4D covariance maps from routinely acquired 3D spectra and facilitates side chain resonance assignments. Thus, we generate two 4D maps from 3D-HcccoNH and 3D-HCcH-TOCSY spectra that each help overcome signal overlap or sensitivity losses. These 4D maps feature HC-HSQCs of individual side chains that can be paired to assigned backbone amide resonances of individual aliphatic signals, and both are obtained from a single modified covariance calculation. Further, we present 4D maps produced using conventional triple resonance experiments to easily assign asparagine side chain amide resonances. The 4D covariance maps encapsulate the lengthy manual pattern recognition used in traditional assignment methods and distill the information as correlations that can be easily visualized. We showcase the utility of the 4D covariance maps with a 10 kDa peptidyl carrier protein and a 52 kDa cyclization domain from a nonribosomal peptide synthetase.

Facilitated structure verification of the biopharmaceutical peptide exenatide by 2D heteronuclear NMR maps.

Exenatide is a peptide based anti-diabetic prescription medication. Until now, the literature has lacked a comprehensive atom-specific molecular characterization for this complex large peptide by NMR spectroscopy that can be effortlessly and rapidly utilized for biopharmaceutical structural veracity. Peptide structure verification by NMR is challenging and cumbersome when reliant on traditional proton-based methodology (through-bond and through-space proton connectivity) alone due to increasing complexity, low signal dispersion, and overlap. These challenges are overcome by using 2D heteronuclear (1H-13C and 1H-15N) maps that not only allow unambiguous signal assignment, but also condense the structural verification information within simplified peptide amide and carbon fingerprint maps. Here we report such simplified amide and carbon fingerprint maps for exenatide; made possible by the first ever comprehensive heteronuclear (1H,13C, and 15N) atom specific assignment of exenatide. These heteronuclear assignments were obtained without any isotopic enrichments i.e. at natural abundance, and hence are easily deployable as routine procedures. Furthermore, we compare the 2D heteronuclear maps of exenatide to a chemically identical peptide differing only in the isomerism of the Cα position of the first amino acid, [dHis1]-exenatide, to demonstrate the uniqueness of these maps. We show that despite deliberate changes in pH, temperature, and concentrations, the differences between the amide maps of exenatide and [dHis1]-exenatide are retained. The work presented here not only provides a facilitated structure verification of exenatide but also a framework for heteronuclear NMR data acquisition and signal assignment of large peptides, at natural abundance, in creating their respective unique 2D fingerprint maps.

Effortless assignment with 4D covariance sequential correlation maps.

Traditional Nuclear Magnetic Resonance (NMR) assignment procedures for proteins rely on preliminary peak-picking to identify and label NMR signals. However, such an approach has severe limitations when signals are erroneously labeled or completely neglected. The consequences are especially grave for proteins with substantial peak overlap, and mistakes can often thwart entire projects. To overcome these limitations, we previously introduced an assignment technique that bypasses traditional pick peaking altogether. Covariance Sequential Correlation Maps (COSCOMs) transform the indirect connectivity information provided by multiple 3D backbone spectra into direct (H, N) to (H, N) correlations. Here, we present an updated method that utilizes a single four-dimensional spectrum rather than a suite of three-dimensional spectra. We demonstrate the advantages of 4D-COSCOMs relative to their 3D counterparts. We introduce improvements accelerating their calculation. We discuss practical considerations affecting their quality. And finally we showcase their utility in the context of a 52 kDa cyclization domain from a non-ribosomal peptide synthetase.

Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps.

Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C(α) and C(ß) separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups.

A 3D time-shared NOESY experiment designed to provide optimal resolution for accurate assignment of NMR distance restraints in large proteins.

Structure determination of proteins by solution NMR has become an established method, but challenges increase steeply with the size of proteins. Notably, spectral crowding and signal overlap impair the analysis of cross-peaks in NOESY spectra that provide distance restraints for structural models. An optimal spectral resolution can alleviate overlap but requires prohibitively long experimental time with existing methods. Here we present a time-shared 3D experiment optimized for large proteins that provides ¹5N and ¹³C dispersed NOESY spectra in a single measurement. NOESY correlations appear in the detected dimension and hence benefit from the highest resolution achievable of all dimensions without increase in experimental time. By design, this experiment is inherently optimal for non-uniform sampling acquisition when compared to current alternatives. Thus, ¹5N and ¹³C dispersed NOESY spectra with ultra-high resolution in all dimensions were acquired in parallel within about 4 days instead of 80 days for a 52 kDa monomeric protein at a concentration of 350 µM.

NMR methods for structural studies of large monomeric and multimeric proteins.

NMR structural studies of large monomeric and multimeric proteins face distinct challenges. In large monomeric proteins, the common occurrence of frequency degeneracies between residues impedes unambiguous assignment of NMR signals. To overcome this barrier, nonuniform sampling (NUS) is used to measure spectra with optimal resolution within reasonable time, new correlation maps resolve previous impasses in assignment strategies, and novel selective methyl labeling schemes provide additional structural probes without cluttering NMR spectra. These advances push the limits of NMR studies of large monomeric proteins. Large multimeric and multidomain proteins are studied by NMR when individual components can also be studied by NMR and have known structures. The structural properties of large assemblies are obtained by identifying binding surfaces, by orienting domains, and employing limited distance constraints. Segmental labeling and the combination of NMR with other methods have helped popularize NMR studies of such systems.

Non-invasive imaging of neuroanatomical structures and neural activation with high-resolution MRI.

Several years ago, manganese-enhanced magnetic resonance imaging (MEMRI) was introduced as a new powerful tool to image active brain areas and to identify neural connections in living, non-human animals. Primarily restricted to studies in rodents and later adapted for bird species, MEMRI has recently been discovered as a useful technique for neuroimaging of invertebrate animals. Using crayfish as a model system, we highlight the advantages of MEMRI over conventional techniques for imaging of small nervous systems. MEMRI can be applied to image invertebrate nervous systems at relatively high spatial resolution, and permits identification of stimulus-evoked neural activation non-invasively. Since the selection of specific imaging parameters is critical for successful in vivo micro-imaging, we present an overview of different experimental conditions that are best suited for invertebrates. We also compare the effects of hardware and software specifications on image quality, and provide detailed descriptions of the steps necessary to prepare animals for successful imaging sessions. Careful consideration of hardware, software, experiments, and specimen preparation will promote a better understanding of this novel technique and facilitate future MEMRI studies in other laboratories.

Thermodynamic profiling of HIV RREIIB RNA-zinc finger interactions.

The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C(2)H(2) zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf beta-sheet. The DeltaC(p) of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in DeltaC(p), as well as DeltaH and DeltaS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.

Cyclic enzymatic solid phase synthesis of isotopically labeled DNA oligonucleotides.

Isotopic labeling of DNA using standard solid phase synthesis requires expensive phosphoramidites that are used in large excess. We have developed a protocol where enzymatic, cyclic, solid phase synthesis of DNA facilitates a more economical use of the less expensive labeled DNA triphosphates (dNTP). In this approach, the DNA template is immobilized on an epoxy-activated solid support. Both the support and the linkage between DNA and resin are inert to high pH conditions which are required for product release in this scheme. Efficient covalent attachment of the DNA to the resin was achieved when the reaction was carried out in MgCl2/CAPS. The enzymatic fill in reaction as well as product release and recycling conditions were optimized for efficient reuse of dNTPs without any purification. The developed protocol was used to generate a selectively [(13)C, (15)N] G labeled 10-mer duplex.

Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins.

The Rev responsive element (RRE), a part of unspliced human immunodeficiency virus (HIV) RNA, serves a crucial role in the production of infectious HIV virions. The viral protein Rev binds to RRE and facilitates transport of mRNA to the cytoplasm. Inhibition of the Rev-RRE interaction disrupts the viral life cycle. Using a phage display protocol, dual zinc finger proteins (ZNFs) were generated that bind specifically to RREIIB at the high affinity Rev binding site. These proteins were further shortened and simplified, and they still retained their RNA binding affinity. The solution structures of ZNF29 and a mutant, ZNF29G29R, have been determined by nuclear magnetic resonance (NMR) spectroscopy. Both proteins form C(2)H(2)-type zinc fingers with essentially identical structures. RNA protein interactions were evaluated quantitatively by isothermal titration calorimetry, which revealed dissociation constants (K(d)'s) in the nanomolar range. The interaction with the RNA is dependent upon the zinc finger structure; in the presence of EDTA, RNA binding is abolished. For both proteins, RNA binding is mediated by the alpha-helical portion of the zinc fingers and target the bulge region of RREIIB-TR. However, ZNF29G29R exhibits significantly stronger binding to the RNA target than ZNF29; this illustrates that the binding of the zinc finger scaffold is amenable to further improvements.