Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab.

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples.

Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies.

Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor.

The structure of the high-affinity nickel-binding site in the Ni,Zn-HypA•UreE2 complex.

The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer.

The role of heat shock proteins in preventing amyloid toxicity.

The oligomerization of monomeric proteins into large, elongated, β-sheet-rich fibril structures (amyloid), which results in toxicity to impacted cells, is highly correlated to increased age. The concomitant decrease of the quality control system, composed of chaperones, ubiquitin-proteasome system and autophagy-lysosomal pathway, has been shown to play an important role in disease development. In the last years an increasing number of studies has been published which focus on chaperones, modulators of protein conformational states, and their effects on preventing amyloid toxicity.

Visualizing the transiently populated closed-state of human HSP90 ATP binding domain.

HSP90 are abundant molecular chaperones, assisting the folding of several hundred client proteins, including substrates involved in tumor growth or neurodegenerative diseases. A complex set of large ATP-driven structural changes occurs during HSP90 functional cycle. However, the existence of such structural rearrangements in apo HSP90 has remained unclear.

NMR assignment of human HSP90 N-terminal domain bound to a long residence time resorcinol ligand.

HSP90 is a major molecular chaperone that helps both folding and stabilization of various client proteins often implicated in growth control and cell survival such as kinases and transcription factors. However, among HSP90 clients are also found numerous oncoproteins and, through its assistance to them, HSP90 has consequently been reported as a promising anticancer target. Several ligand chemotypes, including resorcinol type ligands, were found to inhibit HSP90, most of them in an ATP competitive manner.

Structural basis for the inhibition of IAPP fibril formation by the co-chaperonin prefoldin.

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal prefoldin interfere similarly with the IAPP fibril elongation and secondary nucleation pathways.

Optimized precursor to simplify assignment transfer between backbone resonances and stereospecifically labelled valine and leucine methyl groups: application to human Hsp90 N-terminal domain.

Methyl moieties are highly valuable probes for quantitative NMR studies of large proteins. Hence, their assignment is of the utmost interest to obtain information on both interactions and dynamics of proteins in solution. Here, we present the synthesis of a new precursor that allows connection of leucine and valine pro-S methyl moieties to backbone atoms by linear C-chains.

Backbone and methyl resonances assignment of the 87 kDa prefoldin from Pyrococcus horikoshii.

Prefoldin is a heterohexameric protein assembly which acts as a co-chaperonin for the well conserved Hsp60 chaperonin, present in archaebacteria and the eukaryotic cell cytosol. Prefoldin is a holdase, capturing client proteins and subsequently transferring them to the Hsp60 chamber for refolding. The chaperonin family is implicated in the early stages of protein folding and plays an important role in proteostasis in the cytosol.

In Vitro Production of Perdeuterated Proteins in HO for Biomolecular NMR Studies.

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins.

Spectral editing of intra- and inter-chain methyl-methyl NOEs in protein complexes.

Specific isotopic labeling of methyl groups in a perdeuterated protein background enables the detection of long range NOEs in proteins or high molecular weight complexes. We introduce here an approach, combining an optimized isotopic labeling scheme with a specifically tailored NMR pulse sequence, to distinguish between intramolecular and intermolecular NOE connectivities. In hetero-oligomeric complexes, this strategy enables sign encoding of intra-subunit and inter-subunit NOEs.

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods.

Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific H-C Labeling and Fast Magic-Angle Spinning NMR.

Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme.

Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle.

Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5'-triphosphate (ATP)-dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions.

Solid-State NMR H-N-(C)-H and H-N-C-C 3D/4D Correlation Experiments for Resonance Assignment of Large Proteins.

Solid-state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid-state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of H-detected assignment approaches, which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D).

Observation of CH⋅⋅⋅π Interactions between Methyl and Carbonyl Groups in Proteins.

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long-range "through-space" scalar couplings between methyl and backbone carbonyl groups in proteins.

Unraveling self-assembly pathways of the 468-kDa proteolytic machine TET2.

The spontaneous formation of biological higher-order structures from smaller building blocks, called self-assembly, is a fundamental attribute of life. Although the protein self-assembly is a time-dependent process that occurs at the molecular level, its current understanding originates either from static structures of trapped intermediates or from modeling. Nuclear magnetic resonance (NMR) spectroscopy has the unique ability to monitor structural changes in real time; however, its size limitation and time-resolution constraints remain a challenge when studying the self-assembly of large biological particles.

Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit.

Solid-state NMR spectroscopy allows the characterization of the structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths increase only marginally.

CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.

A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back (13)C-(13)C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu) substantially compromises the efficiency of the TOCSY transfers.

Induced folding in RNA recognition by Arabidopsis thaliana DCL1.

DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA.

Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.

Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic labeling developments is the specific introduction of [(1)H,(13)C]-labeled methyl probes into perdeuterated proteins.

Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs.

Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and V are stereo-specifically labeled.

Probing transient conformational states of proteins by solid-state R(1ρ) relaxation-dispersion NMR spectroscopy.

The function of proteins depends on their ability to sample a variety of states differing in structure and free energy. Deciphering how the various thermally accessible conformations are connected, and understanding their structures and relative energies is crucial in rationalizing protein function. Many biomolecular reactions take place within microseconds to milliseconds, and this timescale is therefore of central functional importance.