The G4C2 hexanucleotide repeat expansion mutation (HREM) in C9ORF72, represents the most common mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Three main disease mechanisms have been proposed to date: C9ORF72 haploinsufficiency, RNA toxicity, and accumulation of dipeptide repeat proteins. Pure GC content of the HREM potentially enables the formation of various non-B DNA structures such as G-quadruplexes and i-motifs. These structures are proposed to act as promoters and regulatory elements affecting replication, transcription and translation of the surrounding region. G-quadruplexes have already been shown on the G-rich sense DNA and RNA strands (G4C2)n, the structure of the anti-sense (G2C4)n strand remains unresolved. Similar C-rich sequences may, under acidic conditions, form i-motifs consisting of two parallel duplexes in a head to tail orientation held together by hemi-protonated C+-C pairs. We show that d(G2C4)n repeats do form i-motif and protonated hairpins even under near-physiological conditions. Rather than forming a DNA duplex, i-motifs persist even in the presence of the sense strand. This preferential formation of G-quadruplex and i-motif/hairpin structures over duplex DNA, may explain HREM replicational and transcriptional instability. Furthermore, i-motifs/hairpins can represent a novel pharmacological target for C9ORF72 associated ALS and FTLD.

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Keywords:monoclonal antibody(s) pH sucrose arginine physical stability protein aggregation protein formulation fluorescence spectroscopy light scattering (dynamic) nuclear magnetic resonance (NMR) spectroscopy a b s t r a c tUnderstanding 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. IntroductionOne fundamental aim during the development of therapeutic proteins is finding formulations that provide sufficient protein stability during long-term storage. Some critical variables of these formulations are solution pH, ionic strength, and the presence of additives. The additives usually belong to the group of sugars, polyols, amino acids, or surfactants. 1,2 Among these, sucrose is the most frequently used in marketed therapeutic protein formulations. 1 From the amino acids, arginine is of considerable interest, as in some cases, it can suppress protein aggregation or reduce the viscosity of highly concentrated protein solutions. 3,4 In addition, the use of different arginine salts is a topic of intense research because the arginine counterion can determine the effect on protein stability. [4][5][6][7] Sucrose and arginine salts can affect the thermal protein unfolding and aggregation differently depending on the protein molecule. The presence of other buffer components, such as NaCl, Abbreviations used: A 2 , second virial coefficient; ACF, autocorrelation function; AF STD , protein-additive saturation transfer difference amplification factors from NMR; D, mutual diffusion coefficient from DLS; DLS, dynamic light scattering; D max , maximum dimension from SAXS; FI350/FI330, intrinsic protein fluorescence intensity ratio 350nm/330nm; IP1, inflection point of the first thermal unfolding (at a lower temperature); IP2, inflection...

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Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein−ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme−ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein−protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.