Low free energy cost of very long loop insertions in proteins

Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 x 10(8) sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.     

Proteolytic sensitivity and helper T-cell epitope immunodominance associated with the mobile loop in Hsp10s.

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.     

Protein redesign.

It has been demonstrated that, for a number of proteins, it is possible to dramatically alter the connectivities between elements of secondary structure. Remarkably large loop insertions are tolerated and many redesigns have generated proteins that successfully fold to stable, active structures. Some redesigns have been entirely the choice of the investigators, whereas others have incorporated a randomization and selection step to identify optimal sequences. These studies have provided basic guidelines for the rational manipulation of protein structure and stability, they have allowed the dissection of folding pathways and they have generated proteins with the potential for practical therapeutic applications.     

Deletional Protein Engineering Based on Stable Fold

Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.     

Classification of protein quaternary structure by functional domain composition

Background  

The number and the arrangement of subunits that form a protein are referred to as quaternary structure. Quaternary structure is an important protein attribute that is closely related to its function. Proteins with quaternary structure are called oligomeric proteins. Oligomeric proteins are involved in various biological processes, such as metabolism, signal transduction, and chromosome replication. Thus, it is highly desirable to develop some computational methods to automatically classify the quaternary structure of proteins from their sequences.     

Prediction of protein quaternary structure with feature selection and analysis based on protein biological features

Information of protein quaternary structure can help to understand the biological functions of proteins. Because wet-lab experiments are both time-consuming and costly, we adopt a novel computational approach to assign proteins into 10 kinds of quaternary structures. By coding each protein using its biochemical and physicochemical properties, feature selection was carried out using Incremental Feature Selection (IFS) method. The thus obtained optimal feature set consisted of 97 features, with which the prediction model was built. As a result, the overall prediction success rate is 74.90% evaluated by Jackknife test, much higher than the overall correct rate of a random guess 10% (1/10). The further feature analysis indicates that protein secondary structure is the most contributed feature in the prediction of protein quaternary structure.     

Exploring the conformational space of protein loops using a mean field technique with MOLS sampling

We have recently developed a computational technique that uses mutually orthogonal Latin square sampling to explore the conformational space of oligopeptides in an exhaustive manner. In this article, we report its use to analyze the conformational spaces of 120 protein loop sequences in proteins, culled from the PDB, having the length ranging from 5 to 10 residues. The force field used did not have any information regarding the sequences or structures that flanked the loop. The results of the analyses show that the native structure of the loop, as found in the PDB falls at one of the low energy points in the conformational landscape of the sequences. Thus, a large portion of the structural determinants of the loop may be considered intrinsic to the sequence, regardless of either adjacent sequences or structures, or the interactions that the atoms of the loop make with other residues in the protein or in neighboring proteins.     

Different strategies to recover the activity of monomeric triosephosphate isomerase by directed evolution

A monomeric version of triosephosphate isomerase from Trypanosoma brucei, MonoTIM, has very low activity, and the same is true for all of the additional monomeric variants so far constructed. Here, we subjected MonoTIM to directed evolution schemes to achieve an activity improvement. The construction of a suitable strain for genetic selection provided an effective way to obtain active catalysts from a diverse population of protein variants. We used this tool to identify active mutants from two different strategies of mutagenesis: random mutagenesis of the whole gene and randomization of loop 2. Both strategies converged in the isolation of mutations Ala43 to Pro and Thr44 to either Ala or Ser, when randomizing the entire gene or to Arg in the case of randomization of loop 2. The kinetic characterization of the two more active mutants showed an increase of 11-fold in k(cat) and a reduction of 4-fold in K(m) for both of them, demonstrating the sensitivity of the selection method. A small difference in growth rate is observed when both mutant genes are compared, which seems to be attributable to a difference in solubility of the expressed proteins.     

In vivo selection of randomly mutated retroviral genomes.

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.     

Structural basis for helper T-cell and antibody epitope immunodominance in bacteriophage T4 Hsp10. Role of disordered loops

Antigen three-dimensional structure potentially limits the access of endoproteolytic processing enzymes to cleavage sites and of class II major histocompatibility antigen-presenting proteins to helper T-cell epitopes. Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli. Promiscuously immunogenic sequences were associated with unstable loops in the three-dimensional structure of T4 Hsp10. The immunodominant sequence lies on the N-terminal flank of the 22-residue mobile loop, which is sensitive to proteolysis in divergent Hsp10s. Several mobile loop deletions that inhibited proteolysis in vitro caused global changes in the helper T-cell epitope map. A mobile loop deletion that strongly stabilized the protein dramatically reduced the immunogenicity of the flanking immunodominant helper T-cell epitope, although the protein retained good overall immunogenicity. Antisera against the mobile loop deletion variants exhibited increased cross-reactivity, most especially the antisera against the strongly stabilized variant. The results support the hypothesis that unstable loops promote the presentation of flanking epitopes and suggest that loop deletion could be a general strategy to increase the breadth and strength of an immune response.     

Expression of synthetic thaumatin genes in yeast

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.     

Sequence structure of van der Waals locks in proteins

Recent works has suggested that proteins in early evolution have gone through a stage of closed loop elements with a typical contour size of 25-35 residues. These closed loops are still the elementary protein units to these days, and can be used to spell out protein sequence/structure relationship through a relatively small number of protein prototypes. In this study we aimed to identify the sequences that are used to lock the loop ends to one another, and to show how an extensive dictionary of such locking pairs can be created using positional correlation data from a large proteome database, and structural data from PDB databases. Such a dictionary can be used in reconstructing the evolutionary pathway the modern proteins have gone through, and in identifying closed loop elements in modern proteins with yet unknown 3D structure.     

Sequence Structure of van der Waals Locks in Proteins

Abstract

Recent works has suggested that proteins in early evolution have gone through a stage of closed loop elements with a typical contour size of 25–35 residues. These closed loops are still the elementary protein units to these days, and can be used to spell out protein sequence/structure relationship through a relatively small number of protein prototypes. In this study we aimed to identify the sequences that are used to lock the loop ends to one another, and to show how an extensive dictionary of such locking pairs can be created using positional correlation data from a large proteome database, and structural data from PDB databases. Such a dictionary can be used in reconstructing the evolutionary pathway the modern proteins have gone through, and in identifying closed loop elements in modern proteins with yet unknown 3D structure.     

RNA recognition site of PP7 coat protein

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.     

An N-terminal protein degradation tag enables robust selection of highly active enzymes

Degradation tags are short peptide sequences that target proteins for destruction by housekeeping proteases. We previously utilized the C-terminal SsrA tag in directed evolution experiments to decrease the intracellular lifetime of a growth-limiting enzyme and thereby facilitate selection of highly active variants. In this study, we examine the N-terminal RepA tag as an alternative degradation signal for laboratory evolution. Although RepA proved to be less effective than SsrA at lowering protein concentrations in the cell, its N-terminal location dramatically reduced the occurrence of truncation and frameshift artifacts in selection experiments. We exploited this improvement to evolve a topologically redesigned chorismate mutase that is intrinsically disordered but already highly active for the conversion of chorismate to prephenate. After three rounds of mutagenesis and high-stringency selection, a robust and more nativelike variant was obtained that exhibited a catalytic efficiency (k(cat)/K(M) = 84000 M(-1) s(-1)) comparable to that of a natural dimeric chorismate mutase. Because of concomitant increases in catalyst yield, the level of intracellular prephenate production increased approximately 30-fold overall over the course of evolution.     

A "loop entropy reduction" phage-display selection for folded amino acid sequences

As a step toward selecting folded proteins from libraries of randomized sequences, we have designed a 'loop entropy reduction'-based phage-display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable structure with the N and C termini close in space, however, this entropic cost is diminished. The host protein function can, therefore, be used to select folded inserted sequences without relying on specific properties of the inserted sequence. This principle is tested using the IgG binding domain of protein L and the lck SH2 domain as host proteins. The results indicate that the loop entropy reduction screen is capable of discriminating folded from unfolded sequences when the proper host protein and insertion point are chosen.     

Altered Motor Activity of Alternative Splice Variants of the Mammalian Kinesin-3 Protein KIF1B

Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin-3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino-terminal region of the protein. The insertions are located in the loop region containing the lysine-rich cluster, also known as the K-loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule-dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K-loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.     

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

Dynamic dissociating homo-oligomers and the control of protein function

Homo-oligomeric protein assemblies are known to participate in dynamic association/disassociation equilibria under native conditions, thus creating an equilibrium of assembly states. Such quaternary structure equilibria may be influenced in a physiologically significant manner either by covalent modification or by the non-covalent binding of ligands. This review follows the evolution of ideas about homo-oligomeric equilibria through the 20th and into the 21st centuries and the relationship of these equilibria to allosteric regulation by the non-covalent binding of ligands. A dynamic quaternary structure equilibria is described where the dissociated state can have alternate conformations that cannot reassociate to the original multimer; the alternate conformations dictate assembly to functionally distinct alternate multimers of finite stoichiometry. The functional distinction between different assemblies provides a mechanism for allostery. The requirement for dissociation distinguishes this morpheein model of allosteric regulation from the classical MWC concerted and KNF sequential models. These models are described alongside earlier dissociating allosteric models. The identification of proteins that exist as an equilibrium of diverse native quaternary structure assemblies has the potential to define new targets for allosteric modulation with significant consequences for further understanding and/or controlling protein structure and function. Thus, a rationale for identifying proteins that may use the morpheein model of allostery is presented and a selection of proteins for which published data suggests this mechanism may be operative are listed.