Lys Side Chains Research Articles

Covalent Targeting of Histidine Residues with Aryl Fluorosulfates: Application to Mcl-1 BH3 Mimetics.

Covalent drugs provide pharmacodynamic and pharmacokinetic advantages over reversible agents. However, covalent strategies have been developed mostly to target cysteine (Cys) residues, which are rarely found in binding sites. Among other nucleophilic residues that could be in principle used for the design of covalent drugs, histidine (His) has not been given proper attention despite being in principle an attractive residue to pursue but underexplored. Aryl fluorosulfates, a mild electrophile that is very stable in biological media, have been recently identified as possible electrophiles to react with the side chains of Lys; however, limited studies are available on aryl fluorosulfates' ability to target His residues. We demonstrate that proper incorporation of an aryl fluorosulfate juxtaposing the electrophile with a His residue can be used to afford rapid optimizations of His-covalent agents. As an application, we report on His-covalent BH3 mimetics targeting His224 of Mcl-1.

Development of Novel Amino Acids Containing N‐mercaptophenetyl (MPE)‐type Auxiliary and Orthogonal Protecting Groups for Sequential Ligation of Multiple Peptides

AbstractChemoselective peptide ligation using ligation auxiliaries is a powerful methodology to extend the scope of native chemical ligation beyond ligation at Cys. Herein, we report the development of novel amino acids containing an N‐mercaptophenethyl (MPE)‐type auxiliary and protecting groups (i. e., tert‐butyloxycarbonyl (Boc), 1,3‐thiazolidine (Thz), and allyloxycarbonyl (Alloc)). These developed amino acids can be introduced into a peptide via 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, enabling deprotection and native chemical ligation in one pot. The combination of Thz‐type and Alloc‐type acids enables sequential peptide ligation, which is applicable to the synthesis of peptides branched from a Lys side chain. We also found that the multiple N‐MPE groups are efficiently cleaved upon treatment with tris(2‐carboxyethyl)phosphine and morpholine in the presence of SnCl2.

Generation of antibody-drug conjugates by proximity-driven acyl transfer and sortase-mediated ligation.

We report a sortase-based site-specific antibody-drug conjugation strategy, which involves an affinity peptide-directed acyl transfer reaction and sortase-mediated peptide ligation. Through the affinity peptide-mediated acyl transfer reaction, an LPXTG-containing peptide is conjugated to a specific Lys side chain of an antibody. Under the assistance of sortase, a protein drug bearing a GG motif reacts specifically with the LPXTG moiety to produce an antibody-drug conjugate. Our strategy for antibody conjugation can be applied not only to chemically synthesized drugs, but also to biologically expressed proteins, and will provide a new sortase-based strategy for the preparation of antibody-drug conjugates.

Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.

NMR Study of CO2 Capture by Butylamine and Oligopeptide KDDE in Aqueous Solution: Capture Efficiency and Gibbs Free Energy of the Capture Reaction as a Function of pH**

We have been interested in the development of rubisco-based biomimetic systems for reversible CO2 capture from air. Our design of the chemical CO2 capture and release (CCR) system is informed by the understanding of the binding of the activator CO2 (A CO2 ) in rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). The active site consists of the tetrapeptide sequence Lys-Asp-Asp-Glu (or KDDE) and the Lys sidechain amine is responsible for the CO2 capture reaction. We are studying the structural chemistry and the thermodynamics of CO2 capture based on the tetrapeptide CH3 CO-KDDE-NH2 ("KDDE") in aqueous solution to develop rubisco mimetic CCR systems. Here, we report the results of 1 H NMR and 13 C NMR analyses of CO2 capture by butylamine and by KDDE. The carbamylation of butylamine was studied to develop the NMR method and with the protocol established, we were able to quantify the oligopeptide carbamylation at much lower concentration. We performed a pH profile in the multi equilibrium system and measured amine species and carbamic acid/carbamate species by the integration of 1 H NMR signals as a function of pH in the range 8≤pH≤11. The determination of ΔG1 (R) for the reaction R-NH2 +CO2 R-NH-COOH requires the solution of a multi-equilibrium equation system, which accounts for the dissociation constants K2 and K3 controlling carbonate and bicarbonate concentrations, the acid dissociation constant K4 of the conjugated acid of the amine, and the acid dissociation constant K5 of the alkylcarbamic acid. We show how the multi-equilibrium equation system can be solved with the measurements of the daughter/parent ratio X, the knowledge of the pH values, and the initial concentrations [HCO3 - ]0 and [R-NH2 ]0 . For the reaction energies of the carbamylations of butylamine and KDDE, our best values are ΔG1 (Bu)=-1.57 kcal/mol and ΔG1 (KDDE)=-1.17 kcal/mol. Both CO2 capture reactions are modestly exergonic and thereby ensure reversibility in an energy-efficient manner. These results validate the hypothesis that KDDE-type oligopeptides may serve as reversible CCR systems in aqueous solution and guide designs for their improvement.

Thiophene-2,3-Dialdehyde Enables Chemoselective Cyclization on Unprotected Peptides, Proteins, and Phage Displayed Peptides.

Ortho-phthalaldehyde has recently found wide potentials for protein bioconjugation and peptide cyclization. Herein, the second-generation dialdehyde-based peptide cyclization method is reported. The thiophene-2,3-dialdehyde (TDA) reacts specifically with the primary amine (from Lys side chain or peptide N-terminus) and thiol (from Cys side chain) within unprotected peptides to generate a highly stable thieno[2,3-c]pyrrole-bridged cyclic structure, while it does not react with primary amine alone. This reaction is carried out in the aqueous buffer and features tolerance of diverse functionalities, rapid and clean transformation, and operational simplicity. The features allow TDA to be used for protein stapling and phage displayed peptide cyclization.

Electrochemically-Driven Secondary Folding and Assembly of Peptides and Proteins

Reversible electrochemical triggering of the random coil to α-helix conformational transition of polylysine, and secondary folding of reflectin A1, was accomplished at a Pt electrode at potentials < |1| V vs. Ag/AgCl. Direct electroreduction of the N-terminus vs. ε-amino groups in lysine (Lys) sidechains, imidazolium groups of histidine-containing reflectin A1, as well as hydronium reduction and electrolysis, could be easily distinguished and deconvolved using differential pulse voltammetry. Electrochemistry was coupled with in situ UV absorbance, circular dichroism, and dynamic light scattering to dynamically follow the evolution of secondary folding and assembly of polylysine and reflectin at different potentials. Isotope experiments in H2O vs. D2O unequivocally confirm that direct electroreduction of ε-NH3 +/ND3 + groups in Lys sidechains, rather than electrochemically generated pH gradient-induced deprotonation, leads to subsequent α-helix formation in polylysine. The site-selective electrochemistry and optical methodologies to be presented herein can be generalized and extended to interrogate other protonation-sensitive biomolecular systems, and potentially provide access to early intermediates and control over the dynamic structural evolution of peptides and proteins.[1] E. Masquelier, S.P. Liang, L. Sepunaru, D. E. Morse, M. J. Gordon, "Reversible Electrochemical Triggering and Optical Interrogation of Polylysine α-helix formation " Bioelectrochemistry vol. 144, p. 108007, 04/2020 2022, doi: 10.1016/j.bioelechem.2021.108007[2] S. P. Liang, R. Levenson, B. Malady, M. J. Gordon, D. E. Morse, and L. Sepunaru, "Electrochemistry as a surrogate for protein phosphorylation: voltage-controlled assembly of reflectin A1," J R Soc Interface, vol. 17, no. 173, Dec 23 2020, doi: 10.1098/rsif.2020.0774.

Formation of versus Recruitment to RNA-Rich Condensates: Controlling Effects Exerted by Peptide Side Chain Identity.

Liquid-liquid phase separation (LLPS), the spontaneous formation of contiguous liquid phases with distinct compositions, has been long known in chemical systems and more recently recognized as a ubiquitous feature of cell biology. We describe a system involving biologically relevant components, synthetic peptides, and total yeast RNA, that has enabled us to explore factors that underlie phase separation. Coulombic complementarity between a cationic peptide and anionic RNA is necessary but not sufficient for formation of a condensed phase in our system. In addition to a net positive charge, the peptide must present the proper type of cationic moiety. Guanidinium groups, as found in the Arg side chain, support phase separation, but ammonium groups, as found in the Lys side chain, or dimethylguanidinium groups, as found in post-translationally modified Arg side chains, do not support phase separation in our system. However, the cationic groups that do not support phase separation via interaction with RNA can nevertheless enable recruitment to a condensed phase, which reveals that the network of forces governing condensed phase formation can differ from the network of forces governing recruitment to such a phase. We introduce a new method for measuring the concentrations of components in condensed phases based on fluorine-containing additives and 19F NMR.

Improved Handling of Peptide Segments Using Side Chain-Based "Helping Hand" Solubilizing Tools.

Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS)remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.

Reversible electrochemical triggering and optical interrogation of polylysine α-helix formation

Reversible electrochemical triggering of the random coil to α-helix conformational transition of polylysine (Lys10, Lys20, Lys50) was accomplished at a Pt electrode at potentials < |1| V vs. Ag/AgCl. Direct electroreduction of the N-terminus vs ε-amino groups in Lys sidechains, as well as hydronium reduction and electrolysis, could be easily distinguished and deconvolved using differential pulse voltammetry. Electrochemistry was coupled with in situ UV absorbance and circular dichroism spectroscopies to dynamically follow the evolution of α-helix formation at different potentials. Isotope experiments in H2O vs. D2O unequivocally confirm that direct electroreduction of ε-NH3+/ND3+ groups in Lys sidechains, rather than electrochemically generated pH gradient-induced deprotonation, leads to subsequent α-helix formation. The site-selective electrochemistry and optical methodologies presented herein can be generalized and extended to interrogate other protonation-sensitive biomolecular systems, and potentially provide access to early intermediates and control over the dynamic structural evolution of peptides and proteins.

Peptide retention time prediction for peptides with post-translational modifications: N-terminal (α-amine) and lysine (ε-amine) acetylation

Development of a peptide retention prediction model in reversed-phase chromatography is reported for acetylated peptides – both N-terminal (α-) and side chain of Lys (ε-amine) residues. Large-scale proteomic 2D LC-MS analyses of acetylated/non-acetylated tryptic digest of whole human cell lysate have been used to assemble representative retention data sets of 25,000+ modified/non-modified pairs. This allowed elucidating chromatographic behaviour of modified peptides in three different separation modes: high pH reversed-phase, HILIC separation on amide phase (first dimension of 2D) and reversed-phase separation with formic acid as ion-pairing modifier in the second dimension. On average, N-terminal acetylation increases peptide RP retention at acidic pH by 5 Hydrophobicity Index units (% acetonitrile). Acetylation of first lysine adds another 4.1%. The magnitude of the retention shift varies greatly depending on the number of modified amines, peptide length, and N-terminal peptide sequence. Large retention shifts have been observed for peptides with hydrophobic N-termini and specifically peptides carrying sequences characteristic for amphipathic helical structures – all in complete agreement with major sequence-specific features of RP retention mechanism. The utility of the modified Sequence Specific Retention Calculator model has been verified for the in-vivo N-terminally acetylated peptides detected by 2D LC-MS/MS analysis of a yeast tryptic digest. The effect of N-terminal acetylation was also evaluated for six different HILIC columns, strong cation- and strong anion exchange separations using previously acquired 2D LC-MS/MS data.

Cluster formation of initiators as a tool to impose conformational stability to unstructured regions of a protein

Entangled with inherent sensitivity to its environment; proteins, nature’s workhorse performs extraordinary tasks vital for the upkeep of the biological world. Conjugating proteins with polymers has been used as a strategy to exploit these functional capabilities beyond their natural limits. However, the molecular level effects of using initiator compounds are not well understood and left unexploited. In this work, we demonstrate that initiator cluster formation reduces the charge–charge repulsions between Lys side chains, on a model protein, and brings them closer to each other through increased non-polar interactions. Crucially, it is found that initiator clusters imposed more conformational stability to a previously identified highly dynamic unstructured C-terminal region of the model protein, without altering the protein’s global structure-dynamic relationship. The possibility of computationally identified initiator cluster formation has enormous potential to allow experimental techniques, such as X-ray crystallographic, to resolve highly dynamic, unstructured regions of a protein, i.e. intrinsically disordered regions, without altering the protein’s global structure-dynamic relationship and thus its stability and activity. This can prove to be a game changer of the field.

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules.

Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here, we used a combination of targeted and unbiased approaches to look for other cases of “chemical rescue” by naturally occurring small molecules. We report in vivo rescue of hundreds of Saccharomyces cerevisiae mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from “off-target” effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued ∼20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.

Penetrating the Blood-Brain Barrier with New Peptide-Porphyrin Conjugates Having anti-HIV Activity.

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide–drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide–porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.

Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method.

Although both the hydrophobic aliphatic chain and hydrophilic -amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21Lys residues, including the engineered Lys-66 with an unusually low p of 5.6. All of the NMR signals for the 21Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-C,N; ,,,-D]-Lys. The complete set of assigned H, C, and N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the C, C, and N signals for Lys-66, which has the deprotonated -amino group, and the large upfield shifts for the H and C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The C and N chemical shifts of the SNase variant selectively labeled with either [-C;,-D]-Lys or SAIL-Lys, dissolved in HO and DO, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20Lys residues. Namely, the deuterium-induced shifts of the C and N signals depend on the ionization states of the -amino group, i.e., 0.32 ppm for C [ND-NH] vs. 0.21 ppm for C [ND-NH] and 1.1 ppm for N[ND-NH] vs. 1.8 ppm for N[ND-NH]. Since the 1D C NMR spectrum of a protein selectively labeled with [-C;,-D]-Lys shows narrow ( 2 Hz) and well-dispersed C signals, the deuterium-induced shift difference of 0.11 ppm for the protonated and deprotonated -amino groups, which corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the -amino group, the C, C, and N signals for a Lys residue with a deprotonated -amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated -amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated -amino groups at physiological pH levels.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS‐CoV‐2 coronavirus encodes an essential papain‐like protease domain as part of its non‐structural protein (nsp)‐3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin‐like ISG15 protein modifications as well as, with lower activity, Lys48‐linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin‐binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non‐covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self‐processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS‐CoV‐2 infection model.

Dynamics of Ionic Interactions at Protein-Nucleic Acid Interfaces.

ConspectusMolecular association of proteins with nucleic acids is required for many biological processes essential to life. Electrostatic interactions via ion pairs (salt bridges) of nucleic acid phosphates and protein side chains are crucial for proteins to bind to DNA or RNA. Counterions around the macromolecules are also key constituents for the thermodynamics of protein–nucleic acid association. Until recently, there had been only a limited amount of experiment-based information about how ions and ionic moieties behave in biological macromolecular processes. In the past decade, there has been significant progress in quantitative experimental research on ionic interactions with nucleic acids and their complexes with proteins. The highly negatively charged surfaces of DNA and RNA electrostatically attract and condense cations, creating a zone called the ion atmosphere. Recent experimental studies were able to examine and validate theoretical models on ions and their mobility and interactions with macromolecules. The ionic interactions are highly dynamic. The counterions rapidly diffuse within the ion atmosphere. Some of the ions are released from the ion atmosphere when proteins bind to nucleic acids, balancing the charge via intermolecular ion pairs of positively charged side chains and negatively charged backbone phosphates. Previously, the release of counterions had been implicated indirectly by the salt-concentration dependence of the association constant.Recently, direct detection of counterion release by NMR spectroscopy has become possible and enabled more accurate and quantitative analysis of the counterion release and its entropic impact on the thermodynamics of protein–nucleic acid association. Recent studies also revealed the dynamic nature of ion pairs of protein side chains and nucleic acid phosphates. These ion pairs undergo transitions between two major states. In one of the major states, the cation and the anion are in direct contact and form hydrogen bonds. In the other major state, the cation and the anion are separated by water. Transitions between these states rapidly occur on a picosecond to nanosecond time scale. When proteins interact with nucleic acids, interfacial arginine (Arg) and lysine (Lys) side chains exhibit considerably different behaviors. Arg side chains show a higher propensity to form rigid contacts with nucleotide bases, whereas Lys side chains tend to be more mobile at the molecular interfaces. The dynamic ionic interactions may facilitate adaptive molecular recognition and play both thermodynamic and kinetic roles in protein–nucleic acid interactions.

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0.

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118 -H1.0 (T118 PKK motif) and T140 -H1.0 (T140 PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118 -H1.0 and pT118 -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/310 helix (residues 119-123), that are almost perpendicular in T118 -H1.0 but have a poorly defined orientation in pT118 -H1.0. Peptides T140 -H1.0 and pT140 -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118 -H1.0 and pT140 -H1.0 has pKa values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.

Hydrophobic Gasket Mutation Produces Proton Selective Gating Pore Currents in Closed Human Voltage-Gated Proton Channels

A ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this apparent “hydrophobic gasket” (HG) when the channel opens. In the human voltage-gated proton channel, hHV1, not only does S4 movement occur during gating, but proton current also flows through this “gating pore” in open channels. To probe the role of the HG in the function of hHV1, we replaced putative HG residues with less hydrophobic residues or acidic Asp.

Unambiguous Identification of Pyroglutamate in Full-Length Biopharmaceutical Monoclonal Antibodies by NMR Spectroscopy.

Biotherapeutic proteins are an indispensable class of pharmaceuticals that present a high degree of structural complexity and are prone to chemical modifications during production, processing, and storage, which have to be tightly controlled. Pyroglutamate (pGlu), a cyclization product of N-terminal Gln or Glu residues, is a widespread post-translational modification in proteins, including monoclonal antibodies (mAbs). The unambiguous identification and quantification of this modification in proteins is challenging, since the mass difference of -17 Da or -18 Da, when formed from Gln or Glu, respectively, is not unique. Moreover, deamidation and dehydration occur not only during cyclization to pGlu, but also during other reactions leading to different types of modifications, like succinimide or isopeptide bond moieties due to cross-linking between Asn or Gln and Lys side chains. Here we report the unambiguous identification and quantification of pGlu in intact mAbs with natural isotope distribution by NMR spectroscopy. The assignment of all 1H, 13C and 15N random coil chemical shifts of pGlu in short reference peptides led to the identification of unique chemical shift pairs that are distinct from the random coil chemical shifts of the natural amino-acid residues. These characteristic correlations are suited for the detection of pGlu in denatured proteins. We achieved complete denaturation of mAbs using a straightforward protocol, and could detect and quantify pGlu, in agreement with available mass spectrometric data. The application to the mAbs rituximab and adalimumab illustrates the potential of our approach for the characterization of biotherapeutics containing isotopes at natural abundance.