Uniquely folded mini-protein motifs.

Mini-proteins containing fewer than 40 amino acids provide simple model systems for studying protein folding and stability as well as serving as scaffolds for the rational design of new functional motifs. This article reviews current progress on the design and characterization of discretely folded mini-protein motifs.     

Ab initio folding simulation of the Trp-cage mini-protein approaches NMR resolution

Here, we report a 100 ns molecular dynamics simulation of the folding process of a recently designed autonomous-folding mini-protein designated as tc5b with a new AMBER force field parameter set developed based on condensed-phase quantum mechanical calculations and a Generalized Born continuum solvent model. Starting from its fully extended conformation, our simulation has produced a final structure resembling that of NMR native structure to within 1A main-chain root mean square deviation. Remarkably, the simulated structure stayed in the native state for most part of the simulation after it reached the state. Of greater significance is that our simulation has not only reached the correct main-chain conformation, but also a very high degree of accuracy in side-chain packing conformation. This feat has traditionally been a challenge for ab initio simulation studies. In addition to characterization of the trajectory, comparison of our results to experimental data is also presented. Analysis of the trajectory suggests that the rate-limiting step of folding of this mini-protein is the packing of the Trp side-chain.     

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation.

A globular domain can be decomposed into compact modules consisting of contiguous 10-30 amino acid residues. The correlation between modules and exons observed in different proteins suggests that each module was encoded by an ancestral exon and that modules were combined into globular domains by exon fusion. Barnase is a single domain RNase consisting of 110 amino acid residues and was decomposed into six modules. We designed a mini-protein by removing the second module, M2, from barnase in order to gain an insight into the structural and functional roles of the module. In the molecular modeling of the mini-protein, we evaluated thermodynamic stability and aqueous solubility together with mechanical stability of the model. We chemically synthesized a mini-barnase with (15)N-labeling at 10 residues, whose corresponding residues in barnase are all found in the region around the hydrophobic core. Circular dichroism and NMR measurements revealed that mini-barnase takes a non-random specific conformation that has a similar hydrophobic core structure to that of barnase. This result, that a module could be deleted without altering the structure of core region of barnase, supports the view that modules act as the building blocks of protein design.     

Design of a soluble mini-protein through tandem duplication of the minimally engineered beta hairpin 'tongue' motif of alpha-hemolysin

In an attempt to fashion a globular protein out of two conjoined beta hairpin structural motif(s), we created a gene encoding, in tandem, two copies of the 40 residues-long transmembrane beta hairpin tongue (BHT) motif of the pore-forming toxin, alpha-hemolysin, of Staphylococcus aureus. Seven selected hydrophobic residues on each copy of the BHT motif's lipid-facing surface were mutated to hydrophilic residues, to prevent or reduce any non-specific aggregation based on hydrophobic interactions. Tandem BHT turned out to be expressed as a soluble polypeptide which could be raised to concentrations of approximately 2mg/ml. It displayed several characteristics of a folded mini-protein, although not the characteristics of a typical well-folded globular protein. These characteristics include (i) far-UV CD and FTIR spectra indicative of the presence of sheet structure mixed with polyproline type II secondary structure, (ii) a near-UV CD spectrum, indicating some formation of tertiary structure, (iii) evidence of unfolding and dissociation transitions in the presence of denaturants, accompanied by increase in random coil content, and (iv) the ability to transform from sheet to helical structure through a biphasic structural transition in the presence of the cosolvent, trifluorethanol. Importantly, however, tandem BHT displayed no cooperativity during unfolding; taken together with the poor structural content revealed in the far-UV CD spectrum and some non-canonical gel filtration behavior seen in the presence of denaturants, this suggests a partially unsuccessful instance of protein design.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

A Second Look at Mini-Protein Stability: Analysis of FSD-1 Using Circular Dichroism, Differential Scanning Calorimetry, and Simulations

Mini-proteins that contain <50 amino acids often serve as model systems for studying protein folding because their small size makes long timescale simulations possible. However, not all mini-proteins are created equal. The stability and structure of FSD-1, a 28-residue mini-protein that adopted the ββα zinc-finger motif independent of zinc binding, was investigated using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The broad melting transition of FSD-1, similar to that of a helix-to-coil transition, was observed by using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The N-terminal β-hairpin was found to be flexible. The FSD-1 apparent melting temperature of 41°C may be a reflection of the melting of its α-helical segment instead of the entire protein. Thus, despite its attractiveness due to small size and purposefully designed helix, sheet, and turn structures, the status of FSD-1 as a model system for studying protein folding should be reconsidered.     

Recurrent RNA motifs as probes for studying RNA-protein interactions in the ribosome

To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing (15)N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R(2) rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.     

Synthesis of N-Glycopeptides by Convergent Assembly

Electron-donating O-benzylated glycosylamine mono-, di- and trisaccharide representing the reducing terminal of the core structure of N-glycans were incorporated, in anomerically pure from into Leu–Lys–Asn–Gly-Gly–Pro hexapeptide that is a partial structure of the Trp-cage mini-protein by convergent assembly. According to our results acylation of electron-donating O-benzylated glycosylamine with peptide acid under the proposed new reaction conditions led to the formation of glycopeptide in good yield and in anomerically pure form for the first time. This convergent approach allows the synthesis of a series of glycopeptides containing different oligosaccharides without the need to resynthesize the peptide for each individual case.     

Synthesis of N-glycopeptides applying glycoamino Acid building blocks with a combined fmoc/boc strategy

Mono-, di- and trisaccharide representing the reducing terminal of the core structure of N-glycans were incorporated into Leu-Lys-Asn-Gly-Gly-Pro hexapeptide that is a partial structure of the Trp-cage mini-protein by linear assembly. These studies provide evidence that the used combination of Fmoc and Boc strategy and mild conditions result in glycopeptides in high purity and reasonable yield.     

A ribonucleopeptide module for effective conversion of an RNA aptamer to a fluorescent sensor

Ribonucleopeptide (RNP) is a new class of scaffold for modular fluorescent sensors. We report here a short RNA motif that induces an efficient communication between the structural changes associated with the ligand-binding event of RNA aptamer and an optical response of a fluorescent RNP module. An optimized short RNA motif was used as a communication module for the rational design of modular RNP sensors. A modular combination of a GTP-binding RNA aptamer, the short RNA motif and the fluorophore-labeled RNP module afforded a fluorescent GTP sensor that retain the ligand-binding affinity of the parent aptamer.     

Mining protein sequences for motifs.

We use methods from Data Mining and Knowledge Discovery to design an algorithm for detecting motifs in protein sequences. The algorithm assumes that a motif is constituted by the presence of a "good" combination of residues in appropriate locations of the motif. The algorithm attempts to compile such good combinations into a "pattern dictionary" by processing an aligned training set of protein sequences. The dictionary is subsequently used to detect motifs in new protein sequences. Statistical significance of the detection results are ensured by statistically determining the various parameters of the algorithm. Based on this approach, we have implemented a program called GYM. The Helix-Turn-Helix motif was used as a model system on which to test our program. The program was also extended to detect Homeodomain motifs. The detection results for the two motifs compare favorably with existing programs. In addition, the GYM program provides a lot of useful information about a given protein sequence.     

Dual structural requirements for multilineage hematopoietic-suppressive activity of chemokine-derived peptides

Many chemokines have direct suppressive activity in vitro and in vivo on primitive hematopoietic cells. However, few chemokine-derived peptides have shown a significant activity in inhibiting hematopoiesis. Interestingly, a peptide derived from the 34-58 sequence of the CXC chemokine platelet factor 4 (PF4) produced a 30-40% inhibition of proliferation of murine hematopoietic progenitors (CFU-MK, CFU-GM, and BFU-E) in vitro, at concentrations of 30-60-fold lower than PF4. The aim of the present work was to define the structural parameters and motifs involved in conferring biological activity to the peptide PF4(34-58). Both structural predictions and determinations revealed a new helical motif that was further localized between residues 38 and 46. This helix was necessary for binding of the peptide and for permitting the functional DLQ motif at position 54-56 to activate the putative receptor site. Peptides lacking either the helical or the DLQ motif were devoid of inhibitory activity on the hematopoietic progenitors in vitro. However, among inactive peptides, only those having the helical motif counteracted the inhibition induced by the active peptide PF4(34-58). This suggested that the helix might be required for peptide interactions with a putative receptor site, whereas the DLQ motif would be implicated in the activation of this receptor. These results identify for the first time the dual requirements for the design of chemokine-derived peptides with high suppressive activity on hematopoiesis, as well as for the design of molecules with antagonistic action.     

Metal-binding and nuclease activity of an antimicrobial peptide analogue of the salivary histatin 5

The salivary antimicrobial peptide histatin 5 is characterized by its cationic nature, structural flexibility, and the presence of two metal-binding sites (the ATCUN motif and a Zn-binding motif). These properties make this peptide a good model for the design of new drugs of low molecular weight. In this work, we have synthesized and studied a new peptide, an analogue of the histatin 5 named ATCUN-C16, which contains both metal-binding centers. The results show that our 20-residue-derived peptide preserves anticandidal activity and exhibits a higher propensity to assume a stable conformation in a hydrophobic environment than do histatin 5 and the C16 peptide that contains the 16 residues of the C-terminal part of histatin 5, although overall our peptide remains a flexible molecule. ACTUN-C16 was found to bind DNA in a gel retardation assay and to have a nuclease activity in the presence of copper and zinc ions and ascorbate. Its nuclease activity can be attributed to the synergistic action of oxidative and hydrolytic activities due to the Cu-ATCUN complex and to the zinc ion coordination, respectively. The results show a new property of this family of salivary peptides and suggest a novel use of this peptide as a small nuclease and biotechnological tool.     

A Compact Native 24-Residue Supersecondary Structure Derived from the Villin Headpiece Subdomain

Many small proteins fold highly cooperatively in an all-or-none fashion and thus their native states are well protected from thermal fluctuations by an extensive network of interactions across the folded structure. Because protein structures are stabilized by local and nonlocal interactions among distal residues, dissecting individual substructures from the context of folded proteins results in large destabilization and loss of unique three-dimensional structure. Thus, mini-protein substructures can only rarely be derived from natural templates. Here, we describe a compact native 24-residues-long supersecondary structure derived from the hyperstable villin headpiece subdomain consisting of helices 2 and 3 (HP24). Using a combination of experimental techniques, including NMR and small-angle x-ray scattering, as well as all-atom replica exchange molecular-dynamics simulations, we show that a variant with stabilizing substitutions (HP24stab) forms a densely packed and compact conformation. In HP24stab, interactions between helices 2 and 3 are similar to those observed in native folded HP35, and the two helices cooperatively stabilize each other by completing the hydrophobic core lining the central part of HP35. Interestingly, even though the HP24wt fragment shows a more expanded and less structured conformation, NMR and simulations demonstrate a preference for a native-like topology. Thus, the two stabilizing residues in HP24stab shift the energy balance toward the native state, leading to a minimal folding motif.     

Min-21 and min-23, the smallest peptides that fold like a cystine-stabilized beta-sheet motif: design, solution structure, and thermal stability.

Small disulfide-rich proteins provide examples of simple and stable scaffolds for design purposes. The cystine-stabilized beta-sheet (CSB) motif is one such elementary structural motif and is found in many protein families with no evolutionary relationships. In this paper, we present NMR structural studies and stability measurements of two short peptides of 21 and 23 residues that correspond to the isolated CSB motif taken from a 28-residue squash trypsin inhibitor. The two peptides contain two disulfide bridges instead of three for the parent protein, but were shown to fold in a native-like fashion, indicating that the CSB motif can be considered an autonomous folding unit. The 23-residue peptide was truncated at the N-terminus. It has a well-defined conformation close to that of the parent squash inhibitor, and although less stable than the native protein, it still exhibits a high T(m) of about 100 degrees C. We suggest that this peptide is a very good starting building block for engineering new bioactive molecules by grafting different active or recognition sites onto it. The 21-residue peptide was further shortened by removing two residues in the loop connecting the second and third cysteines. This peptide exhibited a less well-defined conformation and is less stable by about 1 kcal mol(-)(1), but it might be useful if a higher flexibility is desired. The lower stability of the 21-residue peptide is supposed to result from inadequate lengths of segments connecting the first three cysteines, thus providing new insights into the structural determinants of the CSB motif.