Chimeric Protein Engineering

Protein stability can be enhanced by the incorporation of non-natural amino acids and semi-rigid peptidomimetics to lower the entropic penalty upon protein folding through preorganization. An example is the incorporation of aminoisobutyric acid (Aib, α-methylalanine) into proteins to restrict the Φ and Ψ backbone angles adjacent to Aib to those associated with helix formation. Reverse-turn analogs were introduced into the sequences of HIV protease and ribonuclease A that enhanced their stability and retained their native enzymatic activity. In this work, a chimeric protein, design_4, was engineered, in silico, by replacing the C-terminal helix of full sequence design protein (FSD-1) with a semi-rigid helix mimetic. Residues 1–16 of FSD-1 was ligated in silico with the N-terminus of a phenylbipyridyl-based helix mimetic to form design_4. The designed chimeric protein was stable and maintained the designed fold in a 100-nanosecond molecular dynamics simulation at 280 K. Its β-hairpin adopted conformations that formed three additional hydrogen bonds. Compared to FSD-1, design_4 contained fewer peptide bonds and internal degrees of freedom; it should, therefore, be more resistant to proteolytic degradation and denaturation.     

Sarcomeric myosin heavy chain is degraded by the proteasome

Cardiac myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The balance between these opposing metabolic processes ultimately determines the number of functional contractile units within each cardiac muscle cell. Although alterations in myofibrillar protein degradation have been shown to contribute to cardiac growth and remodeling, the intracellular proteolytic systems responsible for degrading myofibrillar proteins to their constitutive amino acids are currently unknown. Lactacystin, a recently developed, highly specific proteasome inhibitor, was used in this study to examine the role of the proteasome in myosin heavy chain (MHC) degradation in cultured neonatal rat ventricular myocytes. Cells were treated with growth medium alone or with lactacystin (1-50 microM) for up to 48 h. Lactacystin significantly increased the total protein/DNA ratio and markedly prolonged MHC half-life. Other proteasome inhibitors, namely carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (10 microM) and N-acetyl-L-leucyl-L-leucyl-norleucinal (100 microM), were also effective in suppressing MHC degradation. Lactacystin and other proteasome inhibitors also suppressed the markedly accelerated MHC degradation associated with Ca2+ channel blockade but did not prevent the disassembly and loss of myofibrils that accompanied contractile arrest. Thus, sarcomere disassembly precedes the degradation of MHC, which is at least in part mediated by the proteasome.     

Functional mapping of MHC class II polymorphic residues. The alpha-chain controls the specificity for binding an Ad-versus an A k-restricted peptide and the beta-chain region 65-67 controls T cell recognition but not peptide binding.

MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.     

Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes

Many CTL epitopes of clinical importance, particularly those derived from tumor Ags, display relatively poor MHC binding affinity and stability. Because in vivo immunogenicity, and thus the efficacy of peptide-based vaccines, is thought to be determined by MHC/peptide complex stability, there is a need to develop a simple strategy for enhancing the binding of suboptimal epitopes. Toward this goal, the ability to enhance suboptimal peptides through covalent linkage to beta2-microglobulin (beta2m) was explored. Two suboptimal variants of a high-affinity Db-restricted influenza nucleoprotein peptide were covalently linked, via a polypeptide spacer, to the amino terminus of human beta2m and the recombinant fusion proteins expressed in Escherichia coli. When compared with their uncoupled counterparts, the beta2m-linked epitopes display enhanced MHC stabilization and antigenicity. Thus, tethering epitopes to beta2m provides a simple method for augmenting the biological activity of suboptimal peptides and could be useful in the design of peptide-based vaccines or immunotherapeutics.     

Stability and stabilization of globular proteins in solution

Proteins are multifunctional: their amino acid sequences simultaneously determine folding, function and turnover. Correspondingly, evolution selected for compromises between rigidity (stability) and flexibility (folding/function/degradation), to the result that generally the free energy of stabilization of globular proteins in solution is the equivalent to only a few weak intermolecular interactions. Additional increments may come from extrinsic factors such as ligands or specific compatible solutes. Apart from the enthalpic effects, entropy may play a role by reducing the flexibility (cystine bridges, increased proline content), or by water release from residues buried upon folding and association. Additional quaternary interactions and closer packing are typical characteristics of proteins from thermophiles. In halophiles, protein stability and function are maintained by increased ion binding and glutamic acid content, both allowing the protein inventory to compete for water at high salt. Acidophiles and alkalophiles show neutral intracellular pH; proteins facing the outside extremes of pH possess anomalously high contents in ionizable amino acids. Global comparisons of the amino acid compositions and sequences of proteins from mesophiles and extremophiles did not result in general rules of protein stabilization, even after including complete genome sequences into the search. Obviously, proteins are individuals that optimize internal packing and external solvent interactions by very different mechanisms, each protein in its own way. Strategies deduced from specific ultrastable proteins allow stabilizing point mutations to be predicted.     

Evaluation of the relative stability of liganded versus ligand-free protein conformations using Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space-filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log-likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site-directed mutagenesis in the context of protein engineering.     

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

The regulated turnover of endoplasmic reticulum (ER)–resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture–based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover.     

Mitochondrial protein quality control: implications in ageing

Mitochondria represent both a major source for reactive oxygen species (ROS) production and a target for oxidative macromolecular damage. Increased production of ROS and accumulation of oxidized proteins have been associated with cellular ageing. Protein quality control, also referred as protein maintenance, is very important for the elimination of oxidized proteins through degradation and repair. Chaperone proteins have been implicated in refolding of misfolded proteins while oxidized protein repair is limited to the catalyzed reduction of certain oxidation products of the sulfur-containing amino acids, cysteine and methionine, by specific enzymatic systems. In the mitochondria, oxidation of methionine residues within proteins can be catalytically reversed by the methionine sulfoxide reductases, an ubiquitous enzymatic system that has been implicated both in ageing and protection against oxidative stress. Irreversibly oxidized proteins are targeted to degradation by mitochondrial matrix proteolytic systems such as the Lon protease. The ATP-stimulated Lon protease is believed to play a crucial role in the degradation of oxidized proteins within the mitochondria and age-related declines in the activity and/or expression of this proteolytic system have been previously reported. Age-related impairment of mitochondrial protein maintenance may therefore contribute to the age-associated build-up of oxidized proteins and impairment of mitochondrial redox homeostasis.     

Life and Death of Proteins: A Case Study of Glucose-starved Staphylococcus aureus

The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) proteolysis mainly affected proteins with vegetative functions, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenesis enzymes increased; 2) most proteins were prone to aggregation in the clpP mutant; 3) the absence of ClpP correlated with protein denaturation and oxidative stress responses, deregulation of virulence factors and a CodY repression. We suggest that degradation of redundant, inactive proteins disintegrated from functional complexes and thereby amenable to proteolytic attack is a fundamental cellular process in all organisms to regain nutrients and guarantee protein homeostasis.     

Circular beta-lactamase: stability enhancement by cyclizing the backbone.

We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.     

Improvement of biological activity and proteolytic stability of peptides by coupling with a cyclic peptide

The cyclic moiety of an endothelin antagonist peptide RES-701-1, composed of 10 amino acids with an amide bond between alpha-NH(2) of Gly1 and beta-COOH of Asp9, was coupled to some biologically active peptides aiming to improve their activities and stabilities against proteolytic degradation. Coupling of the cyclic peptide to the N-terminal of RGD-peptides, maximally 4-fold improvement of in vitro activity compared to the original peptide has been achieved. Coupling of it to protein farnesyltransferase inhibiting peptides resulted to improve in vitro activity maximally 3-fold. These peptides coupled with the cyclic peptide also showed enhanced stability against some typical proteases. These results indicate that this cyclic peptide can stabilize the conformations of the peptides coupled to its C-terminus. Coupling of our cyclic peptide is anticipated to be a novel conformational stabilizing method for biologically active peptides, results to improve their activity and stability.     

Premature Translational Termination Products Are Rapidly Degraded Substrates for MHC Class I Presentation

Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.     

Characterization of sequences involved in mediating degradation of ornithine decarboxylase in cells and in reticulocyte lysate

Mouse ornithine decarboxylase is a 461-amino-acid protein that is extremely labile. A set of contiguous in-frame deletions were introduced into its C-terminal hydrophilic region. The resulting mutant proteins were expressed in cos monkey cells using an expression vector based on simian virus 40 (SV40) or by in vitro translation in reticulocyte lysate. The degradation of wild-type and mutant proteins was determined in transfected cos cells and in a degradation system based on reticulocyte lysate. Deletion mutants lacking segments of the C-terminus (amino acids 423-461, 423-435, 436-449 and 449-461) were converted into stable proteins in both experimental systems. The mutant lacking amino acids 295-309 was significantly stabilized in transfected cos cells, but was rapidly degraded in reticulocyte-lysate-based degradation mix. Our results suggest that the carboxyl-terminal region encompassing amino acids 423-461 and perhaps also amino acids 295-309 may constitute a signal recognized by the proteolytic machinery that degrades ornithine decarboxylase.     

Phosphorylation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helical peptides

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.     

Bound peptide-dependent thermal stability of major histocompatibility complex class II molecule I-Ek

We used differential scanning calorimetry to study the thermal denaturation of murine major histocompatibility complex class II, I-E(k), accommodating hemoglobin (Hb) peptide mutants possessing a single amino acid substitution of the chemically conserved amino acids buried in the I-Ek pocket (positions 71 and 73) and exposed to the solvent (position 72). All of the I-Ek-Hb(mut) molecules exhibited greater thermal stability at pH 5.5 than at pH 7.4, as for the I-Ek-Hb(wt) molecule, which can explain the peptide exchange function of MHC II. The thermal stability was strongly dependent on the bound peptide sequences; the I-Ek-Hb(mut) molecules were less stable than the I-Ek-Hb(wt) molecules, in good correlation with the relative affinity of each peptide for I-Ek. This supports the notion that the bound peptide is part of the completely folded MHC II molecule. The thermodynamic parameters for I-Ek-Hb(mut) folding can explain the thermodynamic origin of the stability difference, in correlation with the crystal structural analysis, and the limited contributions of the residues to the overall conformation of the I-Ek-peptide complex. We found a linear relationship between the denaturation temperature and the calorimetric enthalpy change. Thus, although the MHC II-peptide complex could have a diverse thermal stability spectrum, depending on the amino acid sequences of the bound peptides, the conformational perturbations are limited. The variations in the MHC II-peptide complex stability would function in antigen recognition by the T cell receptor by affecting the stability of the MHC II-peptide-T cell receptor ternary complex.     

The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway

MHC class I molecules present precisely cleaved peptides of intracellular proteins on the cell surface. For most antigenic precursors, presentation requires transport of peptide fragments into the ER, but the nature of the cytoplasmic peptides and their chaperones is obscure. By tracking proteolytic intermediates in living cells, we show that intracellular proteolysis yields a mixture of antigenic peptides containing only N-terminal flanking residues for ER transport. Some of these peptides were bound to the group II chaperonin TRiC and were protected from degradation. Destabilization of TRiC by RNA interference inhibited the expression of peptide-loaded MHC I molecules on the cell surface. Thus, the TRiC chaperonin serves a function in protecting proteolytic intermediates in the MHC I antigen processing pathway.     

Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are more stable than other peptides. Such peptides should be overrepresented in the peptidome of MHC class I-associated peptides. HLA-B27 binding peptides use anchor residue R at P2 and, although most amino acids are allowed, particular amino acids are overrepresented at P1, including R and K. We show that such N-terminal dibasic peptides are indeed more efficiently presented by HLA-B27. This suggests that HLA-B27 can present peptides from Ags present in fewer copies than required for successful peptide generation for other MHC class I molecules.     

Mutations in the fusion peptide and adjacent heptad repeat inhibit folding or activity of the Newcastle disease virus fusion protein

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172-14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease virus fusion protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the "a" and "d" positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant proteins expressed on the cell surface were characterized for fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the "a" position interfered with proteolytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven "d" position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these "d" mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most "d" position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal fusion activity. That most of the "d" position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the prefusion form of the protein. We also found that mutations within the fusion peptide, at residue 128, can interfere with folding of the protein, implicating this region in folding of the molecule. No characterized mutation enhanced fusion.