Protein Higher Order Structure Research Articles

Covalent Labeling with Isotopically Encoded Reagents for Faster Structural Analysis of Proteins by Mass Spectrometry

Covalent labeling and mass spectrometry (MS) are increasingly being used to obtain higher-order structure of proteins and protein complexes. Because most covalent labels are relatively large, steps must be taken to ensure the structural integrity of the modified protein during the labeling reactions so that correct structural information can be obtained. Measuring labeling kinetics is a reliable way to ensure that a given labeling reagent does not perturb a protein's structure, but obtaining such kinetic information is time and sample intensive because it requires multiple liquid chromatography (LC)-MS experiments. Here we present a new strategy that uses isotopically encoded labeling reagents to measure labeling kinetics in a single LC-MS experiment. We illustrate this new strategy by labeling solvent-exposed lysine residues with commercially available tandem mass tags. After tandem MS experiments, these tags allow the simultaneous identification of modified sites and determination of the reaction rates at each site in a way that is just as reliable as experiments that involve multiple LC-MS measurements.

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.

Physicochemical and Functional Comparability Between the Proposed Biosimilar Rituximab GP2013 and Originator Rituximab

BackgroundRegulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.ObjectiveTo compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods.MethodsPrimary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D 1H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays.ResultsIntact mass analysis of GP2013 and the heavy and light chains using RP HPLC–ESI–MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab’s functional activities, GP2013 could not be distinguished from originator rituximab.ConclusionGP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

Elevated protein carbonylation and oxidative stress do not affect protein structure and function in the long-living naked-mole rat: A proteomic approach

The ‘oxidative stress theory of aging’ predicts that aging is primarily regulated by progressive accumulation of oxidized macromolecules that cause deleterious effects to cellular homeostasis and induces a decline in physiological function. However, our reports on the detection of higher level of oxidized protein carbonyls in the soluble cellular fractions of long-living rodent naked-mole rats (NMRs, lifespan ∼30yrs) compared to short-lived mice (lifespan ∼3.5yrs) apparently contradicts a key tenet of the oxidative theory. As oxidation often inactivates enzyme function and induces higher-order soluble oligomers, we performed a comprehensive study to measure global protein carbonyl level in different tissues of age-matched NMRs and mice to determine if the traditional concept of oxidation mediated impairment of function and induction of higher-order structures of proteins are upheld in the NMRs. We made three intriguing observations with NMRs proteins: (1) protein carbonyl is significantly elevated across different tissues despite of its exceptional longevity, (2) enzyme function is restored despite of experiencing higher level of protein carbonylation, and (3) enzymes show lesser sensitivity to form higher-order non-reducible oligomers compared to short-living mouse proteins in response to oxidative stress. These observations were made based on the global analysis of protein carbonyl and identification of two heavily carbonylated proteins in the kidney, triosephosphate isomerase (TPI) and cytosolic peroxiredoxin (Prdx1). These un-expected intriguing observations thus strongly suggest that oxidative modification may not be the only criteria for impairment of protein and enzyme function; cellular environment is likely be the critical determining factor in this process and may be the underlying mechanism for exceptional longevity of NMR.

Characterization of Protein Higher Order Structure Using Vibrational Circular Dichroism Spectroscopy

Better understanding of protein higher order structures (HOS) is of major interest to researchers in the field of biotechnology and biopharmaceutics. Monitoring a protein's HOS is crucial towards understanding the impact of molecular conformation on the biotechnological application. In addition, maintaining the HOS is critical for achieving robust processes and developing stable formulations of therapeutic proteins. Loss of HOS contributes to increased aggregation, enhanced immunogenicity and loss of function. Selecting the proper biophysical methods to monitor the secondary and tertiary structures of therapeutic proteins remains the central question in this field. In this study, both Fourier Transform Infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy are employed to characterize the secondary structures of various proteins as a function of temperature and pH. Three proteins with different secondary structures were examined, human serum albumin (HSA), myoglobin, and the monoclonal antibody, ofatumumab. This work demonstrates that VCD is useful technique for monitoring subtle secondary structure changes of protein therapeutics that may occur during processing or handling.

Characterization of Protein Higher Order Structure Using Vibrational Circular Dichroism Spectroscopy

Characterization of Protein Higher Order Structure Using Vibrational Circular Dichroism Spectroscopy

Comparative Signature Diagrams to Evaluate Biophysical Data for Differences in Protein Structure Across Various Formulations

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8-anilino-1-naphthalene-sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high-resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.

Comparison of quantitative spectral similarity analysis methods for protein higher-order structure confirmation

Optical and vibrational spectroscopic techniques are important tools for evaluating secondary and tertiary structures of proteins. These spectroscopic techniques are routinely applied in biopharmaceutical development to elucidate structural characteristics of protein products, to evaluate the impact of processing and storage conditions on product quality, and to assess comparability of a protein product before and after manufacturing changes. Conventionally, the degree of similarity between two spectra has been determined visually. In addition to requiring a significant amount of analyst training and experience, visual inspection of spectra is inherently subjective, and any determination of comparability based on visual analysis of spectra is therefore arbitrary. Here, we discuss a general methodology for evaluating the suitability of numerical methods to calculate spectral similarity, and then we apply the methodology to compare four quantitative spectral similarity methods: the correlation coefficient, area of spectral overlap, derivative correlation algorithm, and spectral difference methods. While the most effective spectral similarity method may depend on the particular application, all four approaches are superior to visual evaluation, and each is suitable for assessing the degree of similarity between spectra.

High throughput characterization of structural differences between closely related proteins in solution

Partitioning of a protein in an aqueous two-phase system (ATPS) is governed by interactions of the protein with aqueous media in the two phases. Here we describe how partitioning of proteins in a set of ATPS of different compositions can be used to quantify differences between 3D structures of closely related proteins. We also provide perspective on practical applications of the technology when comparative analysis of the higher-order structure of proteins is desired.

Terahertz Spectroscopic Analysis of Peptides and Proteins

Spectroscopic analysis using the Terahertz frequencies between 0.1-15 THz (3–500 cm−1) has been underutilised by the biochemistry community but is starting to yield some scientifically interesting information. Analysis of structures from simple molecules like N-methylacetamide, to polyamides, peptides and relatively complex proteins provides different types of information dependant on the molecular size. The absorbance spectrum of small molecules is dominated by individual modes and specific hydrogen bonds, peptide spectra have peaks associated with secondary structure, while protein spectra are dominated by ensembles of hydrogen bonds and/or collective modes. Protein dynamics has been studied using Terahertz spectroscopy using proteins like bacteriorhodopsin, illustrating a potential application where this approach can provide complementary global dynamics information to the current nuclear magnetic resonance and fluorescence-based techniques. Analysis of higher-order protein structures like polyomavirus virus-like particles generate quite different spectra compared to their constituent parts. The presence of an extended hydration layer around proteins, first postulated to explain data generated using p-germanium spectroscopy may present a particularly interesting opportunity to better understand protein’s complex interaction with water and small solutes in an aqueous environment. The practical aspects of Terahertz spectroscopy including sample handling, the use of molecular dynamics simulation and orthogonal experiment design are also discussed.

Conformational Comparability of Factor IX–Fc Fusion Protein, Factor IX, and Purified Fc Fragment in the Absence and Presence of Calcium

A long lasting recombinant factor IX -Fc fusion protein (rFIX-Fc) is being developed for the treatment of hemophilia B and is currently in late stage clinical investigation. By limiting injection frequency and maintaining efficacy, rFIX-Fc shows promise as a new therapeutic option for hemophilia B patients. However, before gaining regulatory approval, rFIX-Fc must undergo rigorous analytical and biological testing, in addition to clinical trials. Included in this testing is the need to understand this protein's higher-order structure and dynamics. In this study, we investigated and compared the biophysical properties of rFIX-Fc, rFIX, and Fc using hydrogen/deuterium exchange mass spectrometry and differential scanning calorimetry. Within the limits of these techniques, our results show that structural comparability exists between rFIX and the FIX region of rFIX-Fc. In addition, changes in the structure and dynamics of both proteins, in response to calcium binding, a requirement for FIX function, are also highly comparable. In the case of Fc and Fc region of rFIX-Fc, conformational comparability is also established. These biophysical results further support the conclusion that fusing an immunoglobulin gamma 1 Fc to rFIX does not significantly alter the higher-order structure of FIX or Fc, Ca binding to FIX, or Fc functionality.

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.

The role of higher-order protein structure in supporting binding by heteroclitic monoclonal antibodies: The monoclonal antibody KIM185 to CD18 also binds C4-binding protein

Heteroclitic monoclonal antibodies are characterized by the ability to bind multiple epitopes with little or no similarity. Such antibodies have been reported earlier, but insight into to the molecular basis of this propensity is limited. Here we report that the KIM185 antibody to human CD18 reacts with the plasma protein C4b-binding protein (C4BP). This was revealed during affinity purification procedures where human serum was incubated with surfaces coated with monoclonal antibodies to CD18. Other monoclonal antibodies to CD18 (KIM127 and TS1/18) showed no such interaction with C4BP. We constructed a sandwich-type time-resolved immunofluorometric assay using KIM185 both as capture and developing antibody. By use of proteolytic fragments of KIM185 and recombinant deletion mutants of C4BP the interaction sites were mapped to the variable region of KIM185 and the oligomerization domain of C4BP, respectively. C4BP is a large oligomeric plasma protein that binds activated complement factor C4b and other endogenous ligands as well as microorganisms. By use of the recent crystallographic data on the structure of CD11c/CD18 and prediction of the secondary structure of the C4BP oligomerization domain, we show that epitopes bound by KIM185 in these proteins are unlikely to share any major structural similarity. However, both antigens may form oligomers that would enable avid binding by the antibody. Our report points to the astonishing ability of heteroclitic antibodies to accommodate the binding of multiple proteins with no or little structural similarity within the confined space of the variable regions.

The Utility of Hydrogen/Deuterium Exchange Mass Spectrometry in Biopharmaceutical Comparability Studies

The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confidant comparability assessments of protein higher-order structures than are currently available within the biopharmaceutical industry.

Inhibitory Effects of Glycerol on Growth and Invasion of Human Oral Cancer Cell Lines

It has been reported that glycerol enhances susceptibility to radiotherapy and chemotherapy by a chemical chaperone-like effect which restores mutated protein higher-order structures of the p53 protein because p53 functions are recovered to induce p53-dependent apoptosis through radiotherapy and chemotherapy. On the other hand, based on in vitro and in vivo experiments glycerol alone has not been considered to inhibit growth and invasion of cancer cell lines. In the present in vitro study, glycerol alone was added to oral cancer cell lines (tongue cancer, cervical lymph node [CLN] metastasis of tongue cancer, mandibular gingival cancer) and a cisplatin (CDDP) resistant cell line. The results showed that glycerol inhibited the growth and invasive capacities of the cancer cell lines concentration-dependently. Further, it was demonstrated that the mechanisms of glycerol for inhibiting growth and invasion of the cancer cell lines are inhibition of G1/S phase transition of the cancer cell lines, apoptosis induction, and an inability of cancer cells to use glycerol as an energy substrate. Clinically, this may suggest that glycerol has a potential inhibitory effect on the growth and invasion of CDDP-resistant oral cancer as well as primary and CLN metastatic lesions of oral cancer.

Physicochemical and Biologic Comparability of a Biosimilar Granulocyte Colony-Stimulating Factor with Its Reference Product

Development of biosimilars requires physicochemical and biologic characterization to show comparability with a reference product. Zarzio (filgrastim) is a biosimilar recombinant human granulocyte colony-stimulating factor (G-CSF) that has been approved in the EU using Neupogen as its reference product. The aim of this study was to compare the drug identity, purity, and bioactivity of Zarzio (300 and 480 μg/0.5 mL solution) with Neupogen, using a broad range of standard and advanced analytical methods. Peptide mapping with UV detection and mass determination, circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and liquid chromatography electrospray ionization (LC-ESI) mass spectrometry were among the analyses used to compare primary and higher-order protein structure. Cation-exchange chromatography (CEX) and reversed-phase high-performance liquid chromatography (RP-HPLC) were used to compare polarity and charge. Biologic characterization included comparison of G-CSF receptor binding affinity by surface plasmon resonance spectroscopy, an in vitro cell proliferation assay, and Western blot immunologic binding. The primary structures of Zarzio and Neupogen were shown to be identical by peptide mapping and other tests. CD and NMR spectroscopy demonstrated that the two products have comparable secondary and tertiary structures. RP-HPLC and other methods showed that the products have similar purity profiles. Comparable affinity with the G-CSF receptor GCSFR/CD114 was obtained using surface plasmon resonance spectroscopy, and comparable in vitro bioactivity was shown in a cell proliferation assay. These results show the physicochemical and biologic comparability of Zarzio and its reference product, Neupogen.

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Far-Infrared Spectroscopy of Protein Higher-Order Structures

Far-infrared (FIR) spectroscopy in the spectral region of 50-450 cm(-1) was used to study a series of protein higher-order structures constructed using β-lactoglobulin and polyomavirus capsid protein VP1. There were marked differences in the spectra for β-lactoglobulin monomer and dimer and between untreated β-lactoglobulin and heat-induced gels formed at neutral pH. Untreated β-lactoglobulin and heat-induced gels formed at acidic pH exhibited little difference in their spectra. Assembly of the quaternary structure of polyomavirus virus-like particles also caused large changes in the FIR spectra. These findings suggest that FIR spectroscopy may prove useful in studying some protein quaternary and higher-order structures. There was evidence of detection of β-lactoglobulin dimerization, intermolecular disulfide bonding in heat-induced neutral gels, and polyomavirus virus-like particle assembly but no evidence that FIR could detect β-lactoglobulin fibrils with their polymeric structure and hydrogen-bonded intermolecular β-pleated sheeting.

Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry.

Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

Supramolecular SNARE assembly precedes hemifusion in SNARE-mediated membrane fusion

Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex facilitates intracellular membrane fusion. A single SNARE complex is thought to be insufficient; multiple copies of SNARE complexes must work cooperatively. However, the mechanism by which such a higher-order SNARE protein structure is assembled is unknown. EPR and fluorescence analyses show that at least three copies of target-membrane SNARE proteins self-assemble through the interaction between the transmembrane domains (TMDs), and this multimeric structure serves as scaffolding for trans-SNARE assembly. SNARE core formation in solution induces oligomerization of the TMDs of vesicle-associated SNAREs in the apposing membrane, transiently forming a supramolecular protein structure spanning two membranes. This higher-order protein intermediate evolves, by involving lipid molecules, to the hemifusion state. Hemifusion is subsequently followed by distal leaflet mixing and formation of the cis-SNARE complex.