Tailoring Encodable Lanthanide‐Binding Tags as MRI Contrast Agents

Lanthanide‐binding tags (LBTs), peptide‐based coexpression tags with high affinity for lanthanide ions, have previously been applied as luminescent probes to provide phasing for structure determination in X‐ray crystallography and to provide restraints for structural refinement and distance information in NMR. The native affinity of LBTs for Gd³⁺ indicates their potential as the basis for engineering of peptide‐based MRI agents. However, the lanthanide coordination state that enhances luminescence and affords tightest binding would not be ideal for applications of LBTs as contrast agents, due to the exclusion of water from the inner coordination sphere. Herein, we use structurally defined LBTs as the starting point for re‐engineering the first coordination shell of the lanthanide ion to provide for high contrast through direct coordination of water to Gd³⁺ (resulting in the single LBT peptide, m‐sLBT). The effectiveness of LBTs as MRI contrast agents was examined in vitro through measurement of binding affinity and proton relaxivity. For imaging applications that require targeted observation, fusion to specific protein partners is desirable. However, a fusion protein comprising a concatenated double LBT (dLBT) as an N‐terminal tag for the model protein ubiquitin had reduced relaxivity compared with the free dLBT peptide. This limitation was overcome by the use of a construct based on the m‐sLBT sequence (q‐dLBT–ubiquitin). The structural basis for the enhanced contrast was examined by comparison of the X‐ray crystal structure of xq‐dLBT–ubiquitin (wherein two tryptophan residues are replaced with serine), to that of dLBT‐ubiquitin. The structure shows that the backbone conformational dynamics of the MRI variant may allow enhanced water exchange. This engineered LBT represents a first step in expanding the current base of specificity‐targeted agents available.     

A powerful combinatorial screen to identify high-affinity terbium(III)-binding peptides

Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.     

Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.     

Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins

The acidic tail of alpha-synuclein (ATSalpha) has been shown to protect the glutathione S-transferase (GST)-ATSalpha fusion protein from environmental stresses, such as heat, pH and metal ions. In this study, we further demonstrated that the introduction of ATSalpha into other proteins, such as dehydrofolate reductase and adiponectin, renders the fusion proteins resistant to heat-induced aggregation and that the acidic tail of beta- or gamma-synuclein can also protect the fusion proteins from heat-induced aggregation. Interestingly, the heat resistance of GST-ATSalpha deletion mutants, which contain shorter peptides derived from the highly charged regions of ATSalpha, was approximately proportional to the number of added Glu/Asp residues. However, the negative charges in the ATSalpha-derived peptides appear insufficient to explain the extreme heat resistance of the fusion proteins, since polyglutamates appeared to be much less effective than the ATSalpha-derived peptides in conferring heat resistance on the fusion proteins. These results suggest that not only the negatively charged residues but also the specific amino acid sequence of ATSalpha play an important role in conferring extreme heat resistance on the fusion proteins. Furthermore, the heat-induced secondary structural changes and thermal inactivation curves of GST-ATSalpha deletion mutants indicated that the introduction of ATSalpha-derived peptides does not significantly affect the intrinsic stability of the fusion proteins.     

A “Quenchergenic” Chemoselective Protein Labeling Strategy

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular “quenchergenic” aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.     

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.     

Photo-crosslinking: An Emerging Chemical Tool for Investigating Molecular Networks in Live Cells

Studying protein–protein interactions (PPIs) is useful for understanding cellular functions and mechanisms. Evaluating these PPIs under conditions as similar as possible to native conditions can be achieved using photo-crosslinking methods because of their on-demand ability to generate reactive species in situ by irradiation with UV light. Various fusion tag, metabolic incorporation, and amber codon suppression approaches using various crosslinkers containing aryl azide, benzophenone, and diazirines have been applied in live cells. Mass spectrometry and immunological techniques are used to identify crosslinked proteins based on their capture transient and context-dependent interactions. Herein we discuss various incorporation methods and crosslinkers that have been used for interactome mapping in live cells.     

SARS-CoV-2 Virus−Host Interaction: Currently Available Structures and Implications of Variant Emergence on Infectivity and Immune Response

Coronavirus disease 19, or COVID-19, is an infection associated with an unprecedented worldwide pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which has led to more than 215 million infected people and more than 4.5 million deaths worldwide. SARS-CoV-2 cell infection is initiated by a densely glycosylated spike (S) protein, a fusion protein, binding human angiotensin converting enzyme 2 (hACE2), that acts as the functional receptor through the receptor binding domain (RBD). In this article, the interaction of hACE2 with the RBD and how fusion is initiated after recognition are explored, as well as how mutations influence infectivity and immune response. Thus, we focused on all structures available in the Protein Data Bank for the interaction between SARS-CoV-2 S protein and hACE2. Specifically, the Delta variant carries particular mutations associated with increased viral fitness through decreased antibody binding, increased RBD affinity and altered protein dynamics. Combining both existing mutations and mutagenesis studies, new potential SARS-CoV-2 variants, harboring advantageous S protein mutations, may be predicted. These include mutations S13I and W152C, decreasing antibody binding, N460K, increasing RDB affinity, or Q498R, positively affecting both properties.     

Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein

A universal method that improves protein stability and evolution has thus far eluded discovery. Recently, however, studies have shown that insertional fusion to a protein chaperone stabilized various target proteins with minimal negative effects. The improved stability was derived from insertion into a hyperthermophilic protein, Pyrococcus furiosus maltodextrin‐binding protein (PfMBP), rather than from changes to the target protein sequence. In this report, by evaluating the thermodynamic and kinetic stability of various inserted β‐lactamase (BLA) homologues, we were able to examine the molecular determinants of stability realized by insertional fusion to PfMBP. Results indicated that enhanced stability and suppressed aggregation of BLA stemmed from enthalpic and entropic mechanisms. This report also suggests that insertional fusion to a stable protein scaffold has the potential to be a useful method for improving protein stability, as well as functional protein evolution.     

Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.     

Negatively charged purification tags for selective anion-exchange recovery

A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.     

Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.     

Chemical Properties Determine Solubility and Stability in βγ-Crystallins of the Eye Lens

βγ-Crystallins are the primary structural and refractive proteins found in the vertebrate eye lens. Because crystallins are not replaced after early eye development, their solubility and stability must be maintained for a lifetime, which is even more remarkable given the high protein concentration in the lens. Aggregation of crystallins caused by mutations or post-translational modifications can reduce crystallin protein stability and alter intermolecular interactions. Common post-translational modifications that can cause age-related cataracts include deamidation, oxidation, and tryptophan derivatization. Metal ion binding can also trigger reduced crystallin solubility through a variety of mechanisms. Interprotein interactions are critical to maintaining lens transparency: crystallins can undergo domain swapping, disulfide bonding, and liquid-liquid phase separation, all of which can cause opacity depending on the context. Important experimental techniques for assessing crystallin conformation in the absence of a high-resolution structure include dye-binding assays, circular dichroism, fluorescence, light scattering, and transition metal FRET.     

Can Modern Molecular Modeling Methods Help Find the Area of Potential Vulnerability of Flaviviruses?

Flaviviruses are single-stranded RNA viruses that have emerged in recent decades and infect up to 400 million people annually, causing a variety of potentially severe pathophysiological processes including hepatitis, encephalitis, hemorrhagic fever, tissues and capillaries damage. The Flaviviridae family is represented by four genera comprising 89 known virus species. There are no effective therapies available against many pathogenic flaviviruses. One of the promising strategies for flavivirus infections prevention and therapy is the use of neutralizing antibodies (NAb) that can disable the virus particles from infecting the host cells. The envelope protein (E protein) of flaviviruses is a three-domain structure that mediates the fusion of viral and host membranes delivering the infectious material. We previously developed and characterized 10H10 mAb which interacts with the E protein of the tick-borne encephalitis virus (TBEV) and many other flaviviruses’ E proteins. The aim of this work was to analyze the structure of E protein binding sites recognized by the 10H10 antibody, which is reactive with different flavivirus species. Here, we present experimental data and 3D modeling indicating that the 10H10 antibody recognizes the amino acid sequence between the two cysteines C92-C116 of the fusion loop (FL) region of flaviviruses’ E proteins. Overall, our results indicate that the antibody-antigen complex can form a rigid or dynamic structure that provides antibody cross reactivity and efficient interaction with the fusion loop of E protein.     

Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase

Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light–oxygen–voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase because both of its termini are involved in catalysis. It is shown herein that it is possible to covalently insert LOV domains into the polymerase protein, while preserving its activity and generating new light-responsive allosteric coupling.     

重组CFP10-PPE68融合蛋白检测结核分枝杆菌感染的初步应用

目的以结核分枝杆菌(Mycobacterium tuberculosis,MTB)RD1区特异性培养滤液蛋白10(culture filtrate pro-tein10,CFP10)与戊糖-5-磷酸-3-差异构象酶68(pentose-5-phosphate-3-epimerase68,PPE68)的融合蛋白CFP10-PPE68作为包被抗原,建立检测结核病患者血清中MTB抗体的间接ELISA法。方法采用亲和层析法纯化重组CFP10-PPE68、CFP10和PPE68蛋白;分别以3种蛋白作为包被抗原,建立检测结核病患者血清中MTB特异性抗体的间接ELISA法;采用方阵滴定法对建立的间接ELISA法的条件进行优化;并以临床诊断为金标准,对间接ELISA法的特异性、敏感性和准确性进行验证。结果纯化的重组CFP10-PPE68融合蛋白纯度约为70%。分别以重组CFP10-PPE68、CFP10和PPE68蛋白作为包被抗原的最佳包被浓度分别为2、4、4μg/ml,血清最佳稀释度均为1∶1 000,酶标二抗最佳稀释度均为1∶5 000。3种蛋白对肺外结核的诊断效果均优于肺结核,且重组CFP10-PPE68融合蛋白检测肺外结核的特异性、敏感性及准确性均最佳;3种蛋白诊断结核病的特异性较高,而敏感性相对较低。结论以重组CFP10-PPE68融合蛋白作为包被抗原建立的间接ELISA法诊断MTB感染具有较高的敏感性和特异性,特别是对难以诊断的肺外结核病的诊断有一定的参考价值。     

High level expression and secretion of Fc-X fusion proteins in mammalian cells

We have developed a general expression system that enhances the production and secretion of proteins in mammalian cells. The protein of interest is expressed as a fusion to a signal peptide and the Fc fragment of immunoglobulin as the N-terminal fusion partner, which can direct the cellular processes into expressing and secreting high levels of many different types of proteins. These include secretory proteins, enzymes and soluble domains of membrane proteins, as well as nuclear and regulatory proteins. Typical expression levels of these proteins from stable cell lines ranged from several to 100 microg/ml in conditioned media. The Fc domain helps to solubilize hydrophobic proteins and provides a handle for easy detection and purification of the fusion proteins; and it can be cleaved off by treatment with protease if desired.      

Glyco-Steroidal Amphiphiles (GSAs) for Membrane Protein Structural Study

Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein isolation is essential for structural study of these bio-macromolecules. Detergents are the most commonly used amphiphilic compounds for stabilizing membrane proteins in solution outside a lipid bilayer. We previously introduced a glyco-diosgenin (GDN) detergent that was shown to be highly effective at stabilizing a wide range of membrane proteins. This steroidal detergent has additionally gained attention due to its compatibility with membrane protein structure study via cryo-EM. However, synthetic inconvenience limits widespread use of GDN in membrane protein study. To improve its synthetic accessibility and to further enhance detergent efficacy for protein stabilization, we designed a new class of glyco-steroid-based detergents using three steroid units: cholestanol, cholesterol and diosgenin. These new detergents were efficiently prepared and showed marked efficacy for protein stabilization in evaluation with a few model membrane proteins including two G protein-coupled receptors. Some new agents were not only superior to a gold standard detergent, DDM (n-dodecyl-β-d -maltoside), but were also more effective than the original GDN at preserving protein integrity long term. These agents represent valuable alternatives to GDN, and are likely to facilitate structural determination of challenging membrane proteins.     

From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants

The study of protein–protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.     

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential “developmental hemostatic mismatch risk” associated with transfusions containing plasma exosomes from adults.