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Surfactants are commonly used in biopharmaceutical formulations to stabilize proteins against aggregation. However, the choice of a suitable surfactant for a particular protein is decided mostly empirically, and their mechanism of action on molecular level is largely unknown. Here we show that a straightforward label-free method, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, can be used to detect protein-surfactant interactions in formulations of a model protein, interferon alpha. We find that polysorbate 20 binds with its fatty acid to interferon, and that the binding is stronger at pH closer to the isoelectric point of the protein. In contrast, we did not detect interactions between poloxamer 407 and interferon alpha. Neither of the two surfactants affected the tertiary structure and the thermal stability of the protein as evident from circular dichroism and nanoDSF measurements. Interestingly, both surfactants inhibited the formation of subvisible particles during long-term storage, but only polysorbate 20 reduced the amount of small soluble aggregates detected by size-exclusion chromatography. This proof-of-principle study demonstrates how STD-NMR can be employed to quickly assess surfactant-protein interactions and support the choice of surfactant in protein formulation.

Photocaging is an attractive strategy to control molecular behaviour, for example, in chemical synthesis, interaction studies or photodynamic therapies. Here, we demonstrate that

Photocaging is an attractive strategy to control molecular behaviour, for example, in chemical synthesis, interaction studies or photodynamic therapies. Here, we demonstrate that in situ illumination by the LED NMRtorch approach enables effective and controlled photocage release with simultaneous monitoring of subsequent reactions by solution NMR spectroscopy.

Lipopolysaccharide (LPS) is a cell-wall component of Gram-negative bacteria which contributes to bacterial toxicity. During processes such as cell division, shedding of outer membrane vesicles, or bacterial cell death, LPS is released into the surrounding media. If such contamination got into the bloodstream, it would induce pro-inflammatory immune responses which can result in sepsis and death. Therefore, detection of LPS is essential in the pharmaceutical and food industries to prevent patients being exposed to LPS. The Limulus Amebocyte Lysate (LAL) assay is the current major assay used by industry to detect and quantify LPS contamination. However, in recent years the phenomenon of Low Endotoxin Recovery (LER) has gained significant scientific attention. The phenomenon describes the inability of LAL assays, in some cases, to detect LPS due to a masking effect caused by interaction with formulation excipients. Although the mechanism of LER has not been fully determined, it is widely thought that the origin of the effect is associated with these interactions perturbing the supramolecular formation of LPS aggregates. Whilst the phenomenon of LER is highly complex and remains to be entirely understood, herein we aim to provide a state-of-the-art review of the ongoing and, at times, controversial topics of LER research. We overview the current understanding of the relationship between LPS structure and toxicity, conditions in which the supramolecular arrangement of LPS can be altered, the hypothesised mechanisms of LER, and discuss the possible risk of masked LPS remaining biologically toxic upon administration to patients.

Liquid-liquid phase separation (LLPS) of protein solutions is increasingly recognised as an important phenomenon in cell biology and biotechnology. However, opalescence and concentration fluctuations render LLPS difficult to study, particularly when characterising the kinetics of the phase transition and layer separation. Here, we demonstrate the use of a probe molecule trifluoroethanol (TFE) to characterise the kinetics of protein LLPS by NMR spectroscopy. The chemical shift and linewidth of the probe molecule are sensitive to local protein concentration, with this sensitivity resulting in different characteristic signals arising from the dense and lean phases. Monitoring of these probe signals by conventional bulk-detection

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. Abbreviations Arginine·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)

We explore the ability of amino acid solutions, of L-Trp, L-Tyr or L-Val, to solvate pristine graphene flakes in an aqueous environment, via atomistic molecular dynamics simulations and experimental characterization. In accord with previous theoretical work, simulations of single amino acid adsorption on graphene predict that L-Trp is most strongly bound, followed by L-Tyr then L-Val. As the number of amino acids is increased in the simulations, steric hindrance at the graphene interface and amino acid clustering (most pronounced for L-Tyr) reduces the efficiency of interaction with graphene. Using atomic force microscopy and UV-vis absorption spectroscopy, we determine that all three amino acid solutions can exfoliate and suspend pristine graphene flakes in water. However, L-Trp and L-Tyr solutions are considerably more effective than L-Val: L-Trp produces the most stable suspensions and thinnest graphene flakes compared to L-Tyr, with a mean thickness of 6.4 nm, and a narrow distribution of diameter with a mean value of 16 nm, commensurate with the width of cell membranes. At high concentration of L-Trp and L-Tyr, there was severe instability of the suspensions, along with agglomeration and precipitation; this reflects clustering of amino acid molecules observed in molecular dynamics simulations. This study indicates the potential of amino acids to exfoliate and suspend pristine graphene as a step towards developing non-toxic graphene-based vehicles for drug delivery and other in vivo applications.

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behaviour of scFv mutants harbouring single or multiple site-specific arginine/lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein-protein interactions were measured for the wild-type and lysine enriched (4RK) scFvs on account of weakened short-ranged attractions and increased excluded volume, in contrast to the arginine enriched (7KR) scFv which demonstrated strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild-type and especially for 7KR. Individually swapping lysine back to arginine in 4KR indicated that the observed aggregation propensity of arginine in denaturing conditions was non-specific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strength and denaturing buffers; molecular modelling identified hydrogen-bonding between the arginine side chain and main chain peptide groups, stabilising the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design, or current amyloid prediction methods. This work therefore suggests a simple method to increase the stability of a biopharmaceutical protein against aggregation.

Protein behavior in complex mixtures, such as biological fluids, is often modeled by simplified buffer systems in solution. Here we have used the recently described differential 19 F labelling approach (with NMR detection) to monitor and compare the solution behaviour of three proteins at once: human serum albumin (HSA), transferrin (TrF), and immunoglobulin G (IgG), both in serum and in buffer. We demonstrate that monitoring three proteins simultaneously and independently in biological fluid is possible, and that the presence of other endogenous components greatly changes the association characteristics of these proteins. For example, in the simplified model buffer system, all three proteins diffuse at a similar rate, while in serum HSA diffuses around three times faster than TrF, and four times faster than IgG. This 19 F NMR approach allows characterization of the behaviour of complex multiprotein systems in their native environment, e. g., in biological fluids.

Here, we applied NMR spectroscopy in combination with chemometrics to quantify the adulteration of fresh coconut water, stretched with water-sugar mixtures. Coconut water was extracted from young Costa Rican coconuts and adulterated with concentrations of various sugar solutions. A total of 45 samples were analysed by 1D proton NMR spectroscopy and chemometrics. Results showed highly sensitive quantification, with a limit of detection of adulteration with sugars of 1.3% and a root-mean-squared error of prediction of 0.58%. Interestingly, we identified a regular drift in the chemical shift and a change in the lineshape of malic acid signals concomitant with increasing levels of adulteration. On further investigation, this was found to originate from changes in the concentration of divalent cations, such as magnesium, within the samples. It can be concluded that 1 H NMR spectroscopy enables accurate quantification for the degree of adulteration in this product, with the added discovery finding that the shift and lineshape of the malic acid signal can be utilised as a potential diagnostic marker for partial substitution of fresh coconut water with extrinsic components such as sugar mixtures.

Biopharmaceutical proteins are important drug therapies in the treatment of a range of diseases. Proteins, such as antibodies (Abs) and peptides, are prone to chemical and physical degradation, particularly at the high concentrations currently sought for subcutaneous injections, and so formulation conditions, including buffers and excipients, must be optimized to minimize such instabilities. Therefore, both the protein and small molecule content of biopharmaceutical formulations and their stability are critical to a treatment's success. However, assessing all aspects of protein and small molecule stability currently requires a large number of analytical techniques, most of which involve sample dilution or other manipulations which may themselves distort sample behavior. Here, we demonstrate the application of 1H nuclear magnetic resonance (NMR) spectroscopy to study both protein and small molecule content and stability in situ in high-concentration (100 mg/mL) Ab formulations. We show that protein degradation (aggregation or fragmentation) can be detected as changes in 1D 1H NMR signal intensity, while apparent relaxation rates are specifically sensitive to Ab fragmentation. Simultaneously, relaxation-filtered spectra reveal the presence and degradation of small molecule components such as excipients, as well as changes in general solution properties, such as pH. 1H NMR spectroscopy can thus provide a holistic overview of biopharmaceutical formulation content and stability, providing a preliminary characterization of degradation and acting as a triaging step to guide further analytical techniques.

Development of peptide therapeutics generally involves screening of excipients that inhibit peptide-peptide interactions, hence aggregation, and improve peptide stability. We used the therapeutic peptide plectasin to develop a fast screening method that combines microscale thermophoresis titration assays and molecular dynamics simulations to relatively rank the excipients with respect to binding affinity and to study key peptide-excipient interaction hotspots on a molecular level, respectively. Additionally, 1H-13C-HSQC NMR titration experiments were performed to validate the fast screening approach. The NMR results are in qualitative agreement with results from the fast screening method demonstrating that this approach can be reliably applied to other peptides and proteins as a fast screening method to relatively rank excipients and predict possible excipient binding sites.

One of the major challenges in formulation development of biopharmaceuticals is improving long-term storage stability, which is often achieved by addition of excipients to the final formulation. Finding the optimal excipient for a given protein is usually done using a trial-and-error approach, due to the lack of general understanding of how excipients work for a particular protein. Previously, preferential interactions (binding or exclusion) of excipients with proteins were postulated as a mechanism explaining diversity in the stabilisation effects. Weak preferential binding is however difficult to quantify experimentally, and the question remains whether the formulation process should seek excipients which preferentially bind with proteins, or not. Here, we apply solution NMR spectroscopy to comprehensively evaluate protein-excipient interactions between therapeutically relevant proteins and commonly used excipients. Additionally, we evaluate the effect of excipients on thermal and colloidal protein stability, on aggregation kinetics and protein storage stability at elevated temperatures. We show that there is a weak negative correlation between the strength of protein-excipient interactions and effect on enhancing protein thermal stability. We found that the overall protein-excipient binding per se can be a poor criterion for choosing excipients enhancing formulation stability. Experiments on diverse set of excipients and test proteins reveal that while excipients affect all of the different aspects of protein stability, the effects are very much protein specific, and care must be taken to avoid apparent generalizations if a smaller dataset is being used.

The ability to monitor the behavior of individual proteins in complex mixtures has many potential uses, ranging from analysis of protein interactions in highly-concentrated solutions, modelling biological fluids or the intracellular environment, to optimizing biopharmaceutical co-formulations. Differential labelling NMR approaches, which tradi-tionally use 15N or 13C isotope incorporation during recombinant expression, are not always practical in cases when endogenous proteins are obtained from an organism, or where the expression system does not allow for efficient labelling, especially for larger proteins. This study proposes differential labelling of proteins by covalent attachment of 19F groups with distinct chemical shifts, giving each protein a unique spectral signature which can be monitored by 19F NMR without signal overlap, even in complex mixtures, and without any interfering signals from the buffer or other unlabeled components. Parameters such as signal intensities, translational diffusion coefficients and transverse relaxation rates, which report on the behaviour of individual proteins in the mixture, can be recorded even for proteins as large as antibodies at a wide range of concentrations.

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., 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.

Understanding the effects of additives on therapeutic protein stability is of paramount importance for obtaining stable formulations. In this work, we apply several high- and medium-throughput methods to study the physical stability of a model monoclonal antibody at pH 5.0 and 6.5 in the presence of sucrose, arginine hydrochloride, and arginine glutamate. In low ionic strength buffer, the addition of salts reduces the antibody colloidal and thermal stability, attributed to screening of electrostatic interactions. The presence of glutamate ion in the arginine salt partially reduces the damaging effect of ionic strength increase. The addition of 280 mM sucrose shifts the thermal protein unfolding to a higher temperature. Arginine salts in the used concentration reduce the relative monomer yield after refolding from urea, whereas sucrose has a favorable effect on antibody refolding. In addition, we show 12-month long-term stability data and observe correlations between thermal protein stability, relative monomer yield after refolding, and monomer loss during storage. The monomer loss during storage is related to protein aggregation and formation of subvisible particles in some of the formulations. This study shows that the effect of commonly used additives on the long-term antibody physical stability can be predicted using orthogonal biophysical measurements.

Plectasin is a small, cysteine-rich peptide antibiotic which belongs to the class of antimicrobial peptides and has potential antibacterial activity against various Gram-positive bacteria. In the current study, the effect of pH and ionic strength (NaCl) on the conformational stability of plectasin variants has been investigated. At all physiochemical conditions, peptide secondary structures are intact throughout simulations. However, flexibility increases with pH because of the change in electrostatics around the distinct anionic tetrapeptide (9-12) stretch. Furthermore, plectasin interactions with NaCl were measured by determining the preferential interaction coefficients, Γ23. Generally, wild-type plectasin has higher preference for sodium ions as 9ASP is mutated in other variants. Overall, the Γ23 trend with pH for the two salt conditions remain the same for all variants predominately having accumulation of sodium ions around 10GLU and 12ASP. Insignificant changes in the overall peptide conformational stability are in agreement with the fact that plectasin has three cystines. Thermodynamic integration molecular dynamics simulations supplemented with nuclear magnetic resonance were employed to determine the degree of involvement of three different cystines to the overall structural integrity of the peptide. Both methods show the same order of cystine reduction and complete unfolding is observed only upon reduction of all cystines.

Protein behavior in complex mixtures, such as biological fluids, is often modeled by simplified buffer systems in solution. Here we have used the recently described differential

Colloidal stability is among the key challenges the pharmaceutical industry faces during production and manufacturing of protein therapeutics. Self-association and aggregation processes can not only impair therapeutic efficacy but also induce immunogenic responses in patients. Aggregation-prone regions (APRs) consisting of hydrophobic patches are commonly identified as the source for colloidal instability and rational strategies to mitigate aggregation propensity often require genetic engineering to eliminate hydrophobic amino acid residues. Here, we investigate cucurbit[7]uril (CB[7]), a water soluble macrocycle able to form host-guest complexes with aromatic amino acid residues, as a potential excipient to mitigate protein aggregation propensity. Two monoclonal antibodies (mAbs), one harbouring an APR and one lacking an APR, were first assessed for their colloidal stability (measured as the translational diffusion coefficient) in the presence and absence of CB[7] using dynamic light scattering (DLS). Due to the presence of a tryptophan residue within the APR, we were able to monitor changes in intrinsic fluorescence in response to increasing concentrations of CB[7]. Isothermal titration calorimetry (ITC) and NMR spectroscopy were then used to characterize the putative host-guest interaction. Our results suggest a stabilizing effect of CB[7] on the aggregation-prone mAb, due to the specific interaction of CB[7] with aromatic amino acid residues located within the APR. This provides a starting point for exploring CB[7] as a candidate excipient for the formulation of aggregation prone mAbs.

Engineered protein scaffolds are an alternative to monoclonal antibodies in research and drug design due to their small size, ease of production, versatility, and specificity for chosen targets. One key consideration when engineering such proteins is retaining the original scaffold structure and stability upon insertion of target-binding loops. SQT is a stefin A derived scaffold protein that was used as a model to study possible problems associated with solution behaviour of such aptamers. We used an SQT variant with AU1 and Myc insertion peptides (SQT-1C) to study the effect of peptide insertions on protein structure and oligomerisation. The X-ray structure of monomeric SQT-1C revealed a cystatin-like fold. Furthermore, we show that SQT-1C readily forms dimers and tetramers in solution. NMR revealed that these oligomers are symmetrical, with inserted loops comprising the interaction interface. Two possible mechanisms of oligomerisation are compared using molecular dynamics simulations, with domain swap oligomerisation being thermodynamically favoured. We show that retained secondary structure upon peptide insertion is not indicative of unaltered 3D structure and solution behaviour. Therefore, additional methods should be employed to comprehensively assess the consequences of peptide insertions in all aptamers, particularly as uncharacterized oligomerisation may alter binding epitope presentation and affect functional efficiency.

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

The TREX complex mediates the passage of bulk cellular mRNA export to the nuclear export factor TAP/NXF1 via the export adaptors ALYREF or UIF, which appear to act in a redundant manner. TREX complex recruitment to nascent RNA is coupled with 5′ capping, splicing and polyadenylation. Therefore to facilitate expression from their intronless genes, herpes viruses have evolved a mechanism to circumvent these cellular controls. Central to this process is a protein from the conserved ICP27 family, which binds viral transcripts and cellular TREX complex components including ALYREF. Here we have identified a novel interaction between HSV-1 ICP27 and an N-terminal domain of UIF in vivo, and used NMR spectroscopy to locate the UIF binding site within an intrinsically disordered region of ICP27. We also characterized the interaction sites of the ICP27 homolog ORF57 from KSHV with UIF and ALYREF using NMR, revealing previously unidentified binding motifs. In both ORF57 and ICP27 the interaction sites for ALYREF and UIF partially overlap, suggestive of mutually exclusive binding. The data provide a map of the binding sites responsible for promoting herpes virus mRNA export, enabling future studies to accurately probe these interactions and reveal the functional consequences for UIF and ALYREF redundancy.

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.

Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.

Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases and should be avoided in the final drug product. Understanding the factors leading to LLPS and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (mAb5), its solubility, and parameters extracted using

The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain.

The aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

Adaptor proteins stimulate the nuclear export of mRNA, but their mechanism of action remains unclear. Here, we show that REF/ALY binds mRNA; but upon formation of a ternary complex with TAP the RNA is transferred from REF to TAP, and overexpression of TAP displaces REF from mRNA in vivo . RNA is also handed over from two other adaptors, 9G8 and SRp20 to TAP upon formation of a ternary complex. Interestingly, the RNA-binding affinity of TAP is enhanced 4-fold in vitro once it is complexed with REF. 9G8 and SRp20 also enhance the TAP RNA-binding activity in vitro . Consistent with a model in which TAP directly binds mRNA handed over from adaptors during export, we show that TAP binds mRNA in vivo by an arginine-rich motif in its N-terminal domain. The importance of direct TAP–mRNA interactions is confirmed by the observation that a mutant form of TAP that fails to bind mRNA but retains the ability to bind REF does not function in mRNA export.

WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.

The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~ 400 mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo., Highlights • The effect of Arg·Glu on cell viability was examined with reference to NaCl. • Arg·Glu is of equivalent toxicity to NaCl. • The mechanism of cell death is via apoptosis and is thus not pro-inflammatory. • Arg·Glu as a subcut excipient is not expected to result in atypical inflammation.

Herpesviruses cause life-long infections by evading the host immune system and establishing latent infections. All mammalian herpesviruses express an essential multifunctional protein that is typified by ICP27 encoded by Herpes Simplex Virus 1. The only region that is conserved among the diverse members of the ICP27 family is a predicted globular domain that has been termed the ICP27 homology domain. Here we present the first crystal structure of the ICP27 homology domain, solved to 1.9 Å resolution. The protein is a homo-dimer, adopting a novel intertwined fold with one CHCC zinc-binding site per monomer. The dimerization, which was independently confirmed by SEC-MALS and AUC, is stabilized by an extensive network of intermolecular contacts and a domain-swap involving the two N-terminal helices and C-terminal tails. Each monomer contains a lid motif that can clamp the C-terminal tail of its dimeric binding partner against its globular core, without forming any distinct secondary structure elements. The binding interface was probed with point mutations, none of which had a noticeable effect on dimer formation; however deletion of the C-terminal tail region prevented dimer formation in vivo. The structure provides a template for future biochemical studies and modelling of ICP27 homologs from other herpesviruses.

The sequence-specific RNA-binding proteins SRp20 and 9G8 are the smallest members of the serine- and arginine-rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5'CAUC3'. The SRp20-RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5' cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine-rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip-associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1–155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.

ParG is the prototype of a group of small (

Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.

Tim9 and Tim10 are essential components of the "small Tim" family of proteins that facilitate insertion of polytopic proteins at the inner mitochondrial membrane. The small Tims are themselves imported from the cytosol and are organized in specific translocation assemblies in the intermembrane space. Their conformational properties and how these influence the mechanism of assembly remain poorly understood. Moreover, the three-dimensional structure of the TIM10 complex is unknown. We have characterized the structural properties of these proteins in their free and assembled states using NMR, circular dichroism, and small angle x-ray scattering. We show that the free proteins are largely unfolded in their reduced assembly-incompetent state and molten globules in their oxidized assembly-competent state. Tim10 appears less structured than Tim9 in their respective free oxidized forms and undergoes a larger structural change than Tim9 upon complexation. The NMR data here demonstrates unequivocally that only the oxidized states of the Tim9 and Tim10 proteins are capable of forming a complex. Zinc binding stabilizes the reduced state against proteolysis without significantly affecting the secondary structure. Solution x-ray scattering was used to obtain a molecular envelope for the subunits individually and for their fully functional TIM10 complex. Ab initio shape reconstructions based on the scattering data has allowed us to obtain the first low resolution three-dimensional structure of the TIM10 complex. This is a novel structure that displays extensive surface hydrophobicity. The structure also provides an explanation for the escorting function of this non-ATP-powered chaperone particle.

Summary The ParG protein (8.6 kDa) is an essential component of the DNA partition complex of multidrug resistance plasmid TP228. ParG is a dimer in solution, interacts with DNA sequences upstream of the parFG genes and also with the ParF partition protein both in the absence and presence of target DNA. Here, the solution nuclear magnetic resonance structure of ParG is reported. The ParG dimer is composed of a folded domain formed by two closely intertwined C-terminal parts (residues 33‐76), and two highly mobile tails consisting of N-terminal regions (residues 1‐32). The folded part of ParG has the ribbon‐helix‐helix (RHH) architecture similar to that of the Arc/MetJ superfamily of DNA-binding transcriptional repressors, although the primary sequence similarity is very low. ParG interacts with DNA predominantly via its folded domain; this interaction is coupled with ParG oligomerization. The dimeric RHH structure of ParG suggests that it binds to DNA by inserting the doublestranded b -sheet into the major groove of DNA, in a manner similar to transcriptional repressors from the Arc/MetJ superfamily, and that ParG can function as a transcriptional repressor itself. A new classification of proteins belonging to the Arc/MetJ superfamily and ParG homologues is proposed, based on the location of a conserved positively charged residue at either the beginning or at the end of the b -strand which forms part of the DNA recognition motif.

Graphical abstract, Finding excipients which mitigate protein self-association and aggregation is an important task during formulation. Here, the effect of an equimolar mixture of l-Arg and l-Glu (Arg·Glu) on colloidal and conformational stability of four monoclonal antibodies (mAb1–mAb4) at different pH is explored, with the temperatures of the on-set of aggregation (Tagg) and unfolding (Tm1) measured by static light scattering and intrinsic fluorescence, respectively. Arg·Glu increased the Tagg of all four mAbs in concentration-dependent manner, especially as pH increased to neutral. Arg·Glu also increased Tm1 of the least thermally stable mAb3, but without similar direct effect on the Tm1 of other mAbs. Raising pH itself from 5 to 7 increased Tm1 for all four mAbs. Selected mAb formulations were assessed under accelerated stability conditions for the monomer fraction remaining in solution after storage. The aggregation of mAb3 was suppressed to a greater extent by Arg·Glu than by Arg·HCl. Furthermore, Arg·Glu suppressed the aggregation of mAb1 at neutral pH such that the fraction monomer was near to that at the more typical formulation pH of 5.5. We conclude that Arg·Glu can suppress mAb aggregation with increasing temperature/pH and, importantly, under accelerated stability conditions at weakly acidic to neutral pH.

Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

The new program DASHA is an efficient implementation of common data processing steps for the protein internal dynamic analysis. The “model-free” parameters and their uncertainties (Lipari G., Szabo A.: J. Am. Chem. Soc.104, 4546–4559 (1982) can be calculated from an arbitrary combination of experimental data sets (i.e. heteronuclear1H−15N or1H−13C relaxation times and NOE values at different spectrometer frequencies). Anisotropy of the molecular rotational diffusion could be also taken into account without introduction of the new adjustable parameters into the spectral density functionJ(ω), provided the structure of the molecule is known. Parameters of chemical (conformational) exchange can be estimated from the CPMG spin-lock frequency dependences (Bloomet al.: J. Chem. Phys.42, 1615–1624 (1965); Orekhovet al.: Eur. J. Biochem.219, 887–896 (1994). The program can be used both in the interactive and batch modes. It has sophisticated PostScript plotting facilities.

Major food adulteration and contamination events seem to occur with some regularity, such as the widely publicised adulteration of milk products with melamine and the recent microbial contamination of vegetables across Europe for example. With globalisation and rapid distribution systems, these can have international impacts with far-reaching and sometimes lethal consequences. These events, though potentially global in the modern era, are in fact far from contemporary, and deliberate adulteration of food products is probably as old as the food processing and production systems themselves. This review first introduces some background into these practices, both historically and contemporary, before introducing a range of the technologies currently available for the detection of food adulteration and contamination. These methods include the vibrational spectroscopies: near-infrared, mid-infrared, Raman; NMR spectroscopy, as well as a range of mass spectrometry (MS) techniques, amongst others. This subject area is particularly relevant at this time, as it not only concerns the continuous engagement with food adulterers, but also more recent issues such as food security, bioterrorism and climate change. It is hoped that this introductory overview acts as a springboard for researchers in science, technology, engineering, and industry, in this era of systems-level thinking and interdisciplinary approaches to new and contemporary problems.

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

The spatial structure of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana was determined by two-dimensional 1H-NMR techniques and computational analysis. Nearly complete proton resonance assignments for 61 amino acid residues have been made using two-dimensional (2D) homonuclear total correlated spectroscopy, 2D homonuclear double-quantum-filtered correlated spectroscopy and 2D homonuclear NOE spectroscopy (NOESY) experiments. The cross-peak volumes in NOESY spectra spin-spin coupling constants of vicinal protons NH-C alpha H and C alpha H-C beta H and the observation of slow deuterium exchange of amide protons were used to define local structure and a set of constraints for distance geometry program DIANA. The average root-mean-square deviations are 53 pm for backbone heavy atoms and 118 pm for all heavy atoms of 19 final neurotoxin II conformations. The spatial structure is characterized by a short double-stranded (residues 1-5 and 13-17) and a triple-stranded (residues 22-30, 33-41 and 50-54) antiparallel beta-sheets.

We demonstrate the application of graphene as a support for imaging individual biological molecules in transmission electron microscope (TEM). A simple procedure to produce free-standing graphene membranes has been designed. Such membranes are extremely robust and can support practically any sub-micrometer object. Tobacco mosaic virus has been deposited on graphene samples and observed in a TEM. High contrast has been achieved even though no staining has been applied.