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Abstract We have previously engineered E. coli transketolase (TK) enzyme variants that accept new substrates such as aliphatic or aromatic aldehydes, and also with improved thermal stability. Irreversible aggregation is the primary mechanism of deactivation for TK in the buffers used for biocatalysis, and so we were interested in determining the extent to which this remains true in more complex media, crude cell lysates or even in vivo. Such understanding would better guide future protein engineering efforts. NMR offers a potential approach to probe protein structure changes, aggregation, and diffusion, and19F-labelled amino acids are a useful NMR probe for complex systems with little or no background signal from the rest of the protein or their environment. Here we labelled E. coli TK with two different19F probes, trifluoromethyl-L-phenylalanine (tfm-Phe), and 4-fluoro phenylalanine (4 F-Phe), through site specific non-natural amino acid incorporation. We targeted them to residue K316, a highly solvent exposed site located at the furthest point from the enzyme active sites. Characterisation of the19F-labelled TK variants revealed surprising effects of these mutations on stability, and to some extent on activity. While variant TK-tfm-Phe led to a 7.5 °C increase in the thermal transition midpoint (T m) for denaturation, the TK-4 F-Phe variant largely abolished the aggregation of the enzyme when incubated at 50 °C19. F-NMR revealed different behaviours in response to temperature increases for the two TK variants, displaying opposite temperature gradient chemical shifts, and diverging motion regimes, suggesting that the mutations affected differently both the local environment at this site, and its temperature-induced dynamics. A similar incubation of TK at 40–55 °C is also known to induce higher cofactor-binding affinities, leading to an apparent heat activation under low cofactor concentration conditions. We have hypothesised previously that a heat-inducible conformational change in TK leads to this effect1. H-NMR revealed a temperature-dependent re-structuring of methyl groups, also at 30–50 °C, which may be linked to the heat activation. While our kinetic studies were not expected to observe the heat activation event due to the high cofactor concentrations used, this was not the case for TK-4 F-Phe, which did appear to heat activate slightly at 45 °C. This implied that the mutations at K316 could influence cofactor-binding, despite their location at 47 Å from either active site. Such long-distance effects of mutations are not unprecedented, and indeed we have previously shown how distant mutations can influence active-site loop stability and function in TK, mediated via dynamically coupled networks of residues. Molecular dynamics simulations of the two19F containing variants similarly revealed networks of residues that could couple the changes in dynamics at residue K316, through to changes in active site dynamics. These results independently highlight the sensitivity of active-site function to distant mutations coupled through correlated dynamic networks of residues. They also highlight the potential influence of surface-incorporated probes on protein stability and function, and the need to characterise them well prior to further studies.

In situ illumination of liquid-state NMR samples allows to characterise light-dependent chemical and biological phenomena, but, in practice, the position of an NMR sample deep within the bore of a spectrometer magnet renders such illumination challenging. Here, the authors demonstrate the working principles of a sample illumination device, with an LED array positioned directly at the top of special sample tube, which is inserted into the NMR spectrometer.

Protein liquid-liquid phase separation is an important phenomenon in biology. Here, the authors demonstrate an approach to characterize the evolution of protein phases in both time and space using a fluorinated probe molecule in NMR spectroscopy.

Subcutaneous injection of a low volume (100 mg/mL) formulation is an attractive administration strategy for monoclonal antibodies (mAbs) and other biopharmaceutical proteins. Using concentrated solutions may also be beneficial at various stages of bioprocessing. However, concentrating proteins by conventional techniques, such as ultrafiltration, can be time consuming and challenging. Isolation of the dense fraction produced by macroscopic liquid–liquid phase separation (LLPS) has been suggested as a means to produce high-concentration solutions, but practicality of this method, and the stability of the resulting protein solution have not previously been demonstrated. In this proof-of-concept study, we demonstrate that LLPS can be used to concentrate a mAb solution to >170 mg/mL. We show that the structure of the mAb is not altered by LLPS, and unperturbed mAb is recoverable following dilution of the dense fraction, as judged by 1H nuclear magnetic resonance spectroscopy. Finally, we show that the physical properties and stability of a model high concentration protein formulation obtained from the dense fraction can be improved, for example through the addition of the excipient arginine·glutamate. This results in a stable high-concentration protein formulation with reduced viscosity and no further macroscopic LLPS. Concentrating mAb solutions by LLPS represents a simple and effective technique to progress toward producing high-concentration protein formulations for bioprocessing or administration.AbbreviationsArginine·glutamate (Arg·Glu), Carr-Purcell-Meiboom-Gill (CPMG), critical temperature (TC), high-performance size-exclusion chromatography (HPSEC), liquid–liquid phase separation (LLPS), monoclonal antibody (mAb), nuclear magnetic resonance (NMR), transverse relaxation rate (R2)

ABSTRACT Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo. Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity. IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

ABSTRACT The UL69 protein from human cytomegalovirus (HCMV) is a multifunctional regulatory protein and a member of the ICP27 protein family conserved throughout herpesviruses. UL69 plays many roles during productive infection, including the regulation of viral gene expression, nuclear export of intronless viral RNAs, and control of host cell cycle progression. Throughout the ICP27 protein family, an ability to self-associate is correlated with the functions of these proteins in transactivating certain viral genes. Here, we determined the domain boundaries of a globular ICP27 homology domain of UL69, which mediates self-association, and characterized the oligomeric state of the isolated domain. Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) revealed that residues 200 to 540 form a stable homo-tetramer, whereas a shorter region comprising residues 248 to 536 forms a homo-dimer. Structural analysis of the UL69 tetramer by transmission electron microscopy (TEM) revealed a dimer-of-dimers three-dimensional envelope with bridge features likely from a region of the protein unique to betaherpesviruses. The data provide a structural template for tetramerization and improve our understanding of the structural diversity and features necessary for self-association within UL69 and the ICP27 family. IMPORTANCE Human cytomegalovirus (HCMV) infection is widespread in the human population but typically remains dormant in an asymptomatic latent state. HCMV causes disease in neonates and adults with suppressed or impaired immune function, as the virus is activated into a lytic state. All species of herpesvirus express a protein from the ICP27 family which functions as a posttranscriptional activator in the lytic state. In HCMV, this protein is called UL69. The region of sequence conservation in the ICP27 family is a folded domain that mediates protein interactions, including self-association and functions in transactivation. All members thus far analyzed homo-dimerize, with the exception of UL69, which forms higher-order oligomers. Here, we use biochemical and structural data to reveal that UL69 forms stable tetramers composed of a dimer of dimers and determine a region essential for cross-dimer stabilization.

Raman spectroscopy is emerging as a powerful method for obtaining both quantitative and qualitative information from biological samples. One very interesting area of research, for which the technique has rarely been used, is the detection, quantification and structural analysis of post-translational modifications (PTMs) on proteins. Since Raman spectra can be used to address both of these questions simultaneously, we have developed near infrared Raman spectroscopy with appropriate chemometric approaches (partial least squares regression) to quantify low concentration (4 µM) mixtures of phosphorylated and dephosphorylated bovine αs-casein. In addition, we have used these data in conjunction with Raman optical activity (ROA) spectra and NMR to assess the structural changes that occur upon phosphorylation. [ABSTRACT FROM AUTHOR]

Recrystallization of (E)-5-phenyl-1-(pyridin-2-yl)pent-2-en-4-yn-1-one at room temperature from ethylene glycol in daylight afforded [3,4-bis(phenylethynyl)cyclobutane-1,2-diyl)bis(pyridin-2-ylmethanone], C32H22N2O2 (3), while (E)-5-(4-methylphenyl)-1-(pyridin-2-yl)pent-2-en-4-yn-1-one, C17H13NO (2), remained photoinert. This is the first experimental evidence that pentenynones can be photoreactive when fixed in nearly coplanar parallel positions. During the photoreaction, the bond lengths and angles along the pentenyne chain changed significantly, while the disposition of the pyridyl ring towards the keto group was almost unchanged. The cyclobutane ring adopts an rctt conformation.

Reaction of linear conjugated enynones, 1,5-diarylpent-2-en-4-yn-1-ones [Ar1C≡CCH=CHC(=O)Ar2], with 3-oxo-3-phenylpropanenitrile (NCCH2COPh) in the presence of sodium methoxide MeONa as a base in MeOH at room temperature for 4–26 h affords polyfunctional δ-diketones as a product of regioselective Michael addition to the double carbon–carbon bond of starting enynones. The δ-diketones have been formed as mixtures of two diastereomers in a ratio of 2.5:1 in good general yields of 53–98%. A synthetic potential of the obtained δ-diketones has been demonstrated by heterocyclization with hydrazine into substututed 5,6-dihydro-4H-1,2-diazepine.

Reaction of linear conjugated enynones, 1,5-diarylpent-2-en-4-yn-1-ones, with malononitrile in the presence of lithium diisopropylamide LDA, as a base, in THF at room temperature for 3–7 h resulted in the formation of the product of dimerization, multisubstituted polyfunctional cyclohexanes, 4-aryl-2,6-bis(arylethynyl)-3-(aryloxomethyl)-4-hydroxycyclohexane-1,1-dicarbonitriles, in yields up to 60%. Varying the reaction conditions by decreasing time and temperature and changing the ratio of starting compounds (enynone and malononitrile) allowed isolating some intermediate compounds, which confirmed a plausible reaction mechanism. The relative stability of possible stereoisomers of such cyclohexanes was estimated by quantum chemical calculations (DFT method). The obtained cyclohexanes were found to possess photoluminescent properties.

In the title compound, C21H22ClNO3, the pentadiene unit is nearly planar [maximum deviation = 0.023 (1) Å], but the carbonyl O atom deviates significantly [by 0.304 (1) Å] from its mean plane, which is twisted with respect to the phenyl and chlorobenzene rings by 71.34 (13) and 46.40 (13)°, respectively. In the crystal, inversion-related molecules are linked by two pairs of O—H...O hydrogen bonds, forming chains propagating along [01-1], enclosing R22(16) and R22(22) ring motifs. The chains are linked via C—H...O hydrogen bonds and C—H...π interactions into a three-dimensional supramolecular architecture.

In the title compound, C24H18N4O4, the plane of the phenyl ring is inclined to those of the toluene ring and the dinitro-substituted benzene ring by 66.96 (19) and 47.06 (18)°, respectively, while the planes of the two benzene rings are inclined to one another by 36.26 (19)°. There is an intramolecular N—H...O hydrogen bond between the NH group and the O atom of a nitro group, forming an S(6) ring motif. In the crystal, molecules are linked by C—H...O hydrogen bonds and C—H...π interactions, forming a three-dimensional network. There are also weak π–π interactions present involving the phenyl ring and the dinitro-substituted benzene ring [inter-centroid distance = 3.741 (2) Å].

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions.

The penta-2,4-dien-1-one fragment of the title compound, C23H17ClOS, is twisted by 20.0 (3)°, as measured by the dihedral angle between the planes of the carbonyl group and its attached C atom and the distant C=C double bond and its attached C atom. The 4-chlorophenyl group forms a dihedral angle of 4.0 (3)° with the adjacent carbonyl group. Conjugation between the phenyl ring and the C=C double bond is absent; the dihedral angle between the phenyl ring and the C—C=C fragment is 34.3 (2)°. In the crystal, molecules are linked via C—H...O hydrogen bonds, forming chains parallel to the b-axis direction.

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.