Characterization of conformational heterogeneity via higher-dimensionality, proton-detected solid-state NMR

Site-specific heterogeneity of solid protein samples can be exploited as valuable information to answer biological questions ranging from thermodynamic properties determining fibril formation to protein folding and conformational stability upon stress. In particular, for proteins of increasing molecular weight, however, site-resolved assessment without residue-specific labeling is challenging using established methodology, which tends to rely on carbon-detected 2D correlations. Here we develop purely chemical-shift-based approaches for assessment of relative conformational heterogeneity that allows identification of each residue via four chemical-shift dimensions. High dimensionality diminishes the probability of peak overlap in the presence of multiple, heterogeneously broadened resonances. Utilizing backbone dihedral-angle reconstruction from individual contributions to the peak shape either via suitably adapted prediction routines or direct association with a relational database, the methods may in future studies afford assessment of site-specific heterogeneity of proteins without site-specific labeling

Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state

The active site of a prototypical “rigid” drug target is marked by extensive conformational dynamics

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off-target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly “rigid protein” that substantial active-site dynamics have generally remained unrecognized, despite thousands of medicinal-chemistry studies on this model over decades. Comparing cryogenic X-ray structures, solid-state NMR on micro-crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational-exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines

Solid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: A well-ordered β-sheet core amidst structural heterogeneity.

GrEASy fibrils: Hydrophobins are fungal proteins that assemble into an amphipathic fibrillar monolayer with amyloid properties and a hydrophobic face as water-resistant as Teflon. Solid-state NMR studies on EAS hydrophobin fibrils reveal direct evidence of a partial molecular rearrangement on assembly and an ordered β-sheet-rich core in the context of a whole protein in this functional amyloid

Access to side-chain carbon information in deuterated solids under fast MAS through non-rotor-synchronized mixing.

We demonstrate the accessibility of aliphatic 13C side chain chemical shift sets for solid-state NMR despite perdeuteration and fast MAS using isotropic, non-rotor-synchronized 13C-13C mixing. Combined with amide proton detection, we unambiguously and sensitively detect whole side chain to backbone correlations for two proteins using around 1 mg of sample

Frustrated flexibility in metal-organic frameworks

Stimuli-responsive flexible metal-organic frameworks (MOFs) remain at the forefront of porous materials research due to their enormous potential for various technological applications. Here, we introduce the concept of frustrated flexibility in MOFs, which arises from an incompatibility of intra-framework dispersion forces with the geometrical constraints of the inorganic building units. Controlled by appropriate linker functionalization with dispersion energy donating alkoxy groups, this approach results in a series of MOFs exhibiting a new type of guest- and temperature-responsive structural flexibility characterized by reversible loss and recovery of crystalline order under full retention of framework connectivity and topology. The stimuli-dependent phase change of the frustrated MOFs involves non-correlated deformations of their inorganic building unit, as probed by a combination of global and local structure techniques together with computer simulations. Frustrated flexibility may be a common phenomenon in MOF structures, which are commonly regarded as rigid, and thus may be of crucial importance for the performance of these materials in various applications

Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 100 TCID50 MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10−5 TCID50 MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue