Contemporary strategies for the stabilization of peptides in the alpha-helical conformation

Herein we review contemporary synthetic and protein design strategies to stabilize the alpha-helical motif in short peptides and miniature proteins. Advances in organometallic catalyst design, specifically for the olefin metathesis reaction, enable the use of hydrocarbon bridges to either crosslink side chains of specific residues or mimic intramolecular hydrogen bonds with carbon-carbon bonds. The resulting hydrocarbon-stapled and hydrogen bond surrogate alpha-helices provide unique synthetic ligands for targeting biomolecules. In the protein design realm, several classes of miniature proteins that display stable helical domains have been engineered and manipulated with powerful in vitro selection technologies to yield libraries of sequences that retain their helical folds. Rational re-design of these scaffolds provide distinctive reagents for the modulation of protein-protein interactions.     

In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner

Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.     

LRP130, a pentatricopeptide motif protein with a noncanonical RNA-binding domain,is bound in vivo to mitochondrial and nuclear RNAs

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.     

Preparation of 3D fibrin scaffolds for stem cell culture applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.     

Effective induction of anti-melanoma immunity following genetic vaccination with synthetic mRNA coding for the fusion protein EGFP.TRP2

RNA-based genetic immunization represents an alternative novel strategy for antigen-specific cancer vaccines. In the present paper we investigate the use of synthetic messenger RNA in an experimental melanoma model. We show that gene gun-based immunization using synthetic RNA mediates gene expression in the epidermis and effectively induces antigen-specific cellular and humoral immunity in mice in vivo. Importantly, bombardment of the skin with RNA coding for the melanocytic self-antigen TRP2 linked to the immunogenic protein EGFP was associated with protection against experimentally induced B16 melanoma lung metastases and vitiligo-like fur depigmentation. Our results provide a scientific basis for clinical trials using synthetic mRNA encoding melanocytic antigens linked to immunogenic helper proteins for vaccination of patients with melanoma. Julia Steitz and Cedrik M. Britten contributed equally to this work.     

Alternative non-antibody scaffolds for molecular recognition

Originally proposed one decade ago, the idea of engineering proteins outside the immunoglobulin family for novel binding functions has evolved as a powerful technology. Several classes of protein scaffolds proved to yield reagents with specificities and affinities in a range that was previously considered unique to antibodies. Such engineered protein scaffolds are usually obtained by designing a random library with mutagenesis focused at a loop region or at an otherwise permissible surface area and by selection of variants against a given target via phage display or related techniques. Whereas a plethora of protein scaffolds has meanwhile been proposed, only few of them were actually demonstrated to yield specificities towards different kinds of targets and to offer practical benefits such as robustness, smaller size, and ease of expression that justify their use as a true alternative to conventional antibodies or their recombinant fragments. Currently, the most promising scaffolds with broader applicability are protein A, the lipocalins, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin. Corresponding binding proteins are not only of interest as research reagents or for separation in biotechnology but also as potential biopharmaceuticals, especially in the areas of cancer, autoimmune and infectious diseases as well as for in vivo diagnostics. The medical prospects have boosted high commercial expectations, and many of the promising scaffolds are under development by biotech start-up companies. Although some issues still have to be addressed, for example immunogenicity, effector functions, and plasma half-life in the context of therapeutic use or low-cost high-throughput selection for applications in proteomics research, it has become clear that scaffold-derived binding proteins will play an increasing role in biotechnology and medicine.     

Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach

Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes.     

Protein and RNA engineering to customize microbial molecular reporting

Nature takes advantage of the malleability of protein and RNA sequence and structure to employ these macromolecules as molecular reporters whose conformation and functional roles depend on the presence of a specific ligand (an "effector" molecule). By following nature's example, ligand-responsive proteins and RNA molecules are now routinely engineered and incorporated into customized molecular reporting systems (biosensors). Microbial small-molecule biosensors and endogenous molecular reporters based on these sensing components find a variety of applications that include high-throughput screening of biosynthesis libraries, environmental monitoring, and novel gene regulation in synthetic biology. Here, we review recent advances in engineering small-molecule recognition by proteins and RNA and in coupling in vivo ligand binding to reporter-gene expression or to allosteric activation of a protein conferring a detectable phenotype. Emphasis is placed on microbial screening systems that serve as molecular reporters and facilitate engineering the ligand-binding component to recognize new molecules.     

Kinetics of messenger accumulation coding for IFN gamma, related to modifications in the poly(A) RNA population of activated human lymphocytes.

Exposure of human lymphocytes to a mitogen induces the appearance of newly synthesized RNAs and proteins. This study describes the changes in overall synthesis as measured by pulse labelling of PHA treated lymphocytes as well as a qualitative analysis of the protein synthetic patterns "in vivo" and "in vitro". Both the levels of RNA and protein synthesis increase drastically in PHA stimulated cells, while cultures incubated without mitogen remained at background levels. The low translational activity in control cells is not due to the absence of messengers since the extracted RNAs clearly direct the synthesis of high molecular weight proteins when translated "in vitro". A number of qualitative differences are seen in the "in vitro" translation of RNA extracted from induced and non-induced lymphocytes, although the apparent protein synthetic pattern "in vivo" remains identical. The secretion of IFN- gamma is one of the newly expressed functions in stimulated lymphocytes and therefore has been studied more detailed in a time-course of the messenger level compared to the secreted activity of the medium. A specific probe was used to quantitate in Northern blot's the accumulation of mRNA coding for IFN- gamma.     

An RNA-binding chameleon

The arginine-rich RNA binding motif is found in a wide variety of proteins, including several viral regulatory proteins. Although related at the primary sequence level, arginine-rich domains from different proteins adopt different conformations depending on the RNA site recognized, and in some cases fold only in the context of RNA. Here we show that the RNA binding domain of the Jembrana disease virus (JDV) Tat protein is able to recognize two different TAR RNA sites, from human and bovine immunodeficiency viruses (HIV and BIV, respectively), adopting different conformations in the two RNA contexts and using different amino acids for recognition. In addition to the conformational differences, the JDV domain requires the cyclin T1 protein for high-affinity binding to HIV TAR, but not to BIV TAR. The "chameleon-like" behavior of the JDV Tat RNA binding domain reinforces the concept that RNA molecules can provide structural scaffolds for protein folding, and suggests mechanisms for evolving distinct RNA binding specificities from a single multifunctional domain.     

Examination of potato virus X proteins synthesized in infected tobacco plants.

Nucleotide sequence analysis of potato virus X (PVX) genomic RNA predicts five open reading frames (ORFs). Previous analysis of total RNAs from PVX-infected leaf tissue suggested that six subgenomic RNAs are synthesized during infection. However, the proteins encoded by the genomic RNA, the subgenomic RNAs, or the predicted ORFs have not been identified in vivo. To characterize the coding properties of the viral RNA, particularly to determine whether the five predicted ORFs function in vivo, total protein extracts prepared from PVX-infected leaf tissue were analyzed by using antibodies raised against virus-specific synthetic peptides and against the virus capsid protein. Dot blot analyses showed that these antibodies reacted to PVX-infected extracts, indicating in vivo expression of the five predicted ORFs. In addition, Western blot (immunoblot) analysis of the extracts showed that ORF 1, 2, 3, and 4 peptide antisera and coat protein antiserum detect predominantly a single protein.     

Regulation of protein activity with small-molecule-controlled inteins

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.     

The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing.

The P gene of Sendai virus expresses as many as eight proteins, two of which (V and W) are expressed only from edited mRNAs; only the P protein is known to be involved in RNA synthesis. To examine the functions of the other P gene proteins, we developed an in vivo system in which genome replication is driven by plasmid generated viral proteins. We found that P was essential for this process, whereas V and W were not only non-essential, they were inhibitory. By using various P gene deletions and varying the amounts of plasmids transfected, we provide evidence that P is a modular protein. The N-terminal domain (shared with V and W) binds the L or polymerase protein, whereas the C-terminal domain binds the nucleoprotein NP. A model of paramyxovirus RNA synthesis is presented, and the implications of negative regulation during persistent infection are discussed.     

Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75.

The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.     

A rapid flow cytometry assay for the relative quantification of protein encapsulation into bacterial microcompartments

Bacterial microcompartments (MCPs) are protein‐based organelles that have been suggested as scaffolds for creating in vivo nanobioreactors. One of the key steps towards engineering MCPs is to understand and maximize the encapsulation of enzymes into the lumen of the MCP. However, there are currently no high‐throughput methods for investigating protein encapsulation. Here, we describe the development of a rapid in vivo assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. In this assay, we fuse a degradation peptide to a MCP‐targeted fluorescence reporter and use flow cytometry to measure overall fluorescence from the encapsulated, protected reporter protein. Using this assay, we characterized various MCP‐targeting signal sequence mutants for their ability to encapsulate proteins and identified mutants that encapsulate a greater amount of protein than the wild type signal sequence. This assay is a powerful tool for reporting protein encapsulation and is an important step towards encapsulating metabolic enzymes into MCPs for synthetic biochemical pathways.     

Different complexes are formed on the 3'' end of histone mRNA with nuclear and polyribosomal proteins.

Specific protein-RNA complexes are formed by incubating a synthetic histone mRNA 3' end (a 30 nucleotide stem-loop structure) RNA with extracts of either nuclei or polyribosomes. The complex formed between the stem-loop and nuclear proteins has a lower electrophoretic mobility than the complex formed between the stem-loop and polyribosomal proteins. Binding of the synthetic 3' end by both polyribosomal and nuclear proteins is abolished when two of the conserved uridine residues in the loop are replaced with adenosines. UV crosslinking of the protein complexes to the synthetic RNA resulted in transferring radiolabel to similar sized proteins, 50 kD, in both the nuclear and polyribosomal extracts.     

Monobodies and other synthetic binding proteins for expanding protein science

Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in‐depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.