Solution structure of alpha t alpha, a helical hairpin peptide of de novo design.

alpha t alpha is a 38-residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui Y, Weaver DL Osterhout JJ, 1995, Protein Sci 4:286-295). A previous study of the carboxylate form of alpha t alpha by CD and two-dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675-3679). Here, the solution structure of alpha t alpha as determined by two-dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J-couplings were used. One-hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty-three of the lowest-energy structures consistent with the experimental restraints were analyzed. The results presented here show that alpha t alpha is comprised of two associating helices connected by a turn region.     

De novo ligand design to an ensemble of protein structures

We describe a combinatorial method for de novo ligand design to an ensemble of receptor structures. Receptor conformations, protonation states, and structural water molecules are considered consistently within the framework of de novo ligand design. The method relies on Monte Carlo optimization to search the space of ligand structures, conformations, and rigid-body movements as well as receptor models. The method is applied to an ensemble of HIV protease and human collagenase receptor models. Ligand structures generated de novo exhibit the correct hydrogen-bonding pattern in the core of the active site, with hydrophobic groups extending into the receptor S1 and S1' pocket space. Furthermore, it is shown that known ligands are recovered in the correct binding mode and in the native, most tightly binding receptor model.     

De novo design,synthesis and evaluation of a non-steroidal diphenylnaphthyl propylene ligand for the estrogen receptor

There is still a strong need for additional diversity and new chemical scaffolds to allow for the exploration of improved tissue selectivity and finding better selective estrogen receptor modulators (SERMs). Using a de novo design technology a diphenylnaphthyl propylene scaffold, exemplified by (E)-9b, with ER antagonist activity has been generated. It was prepared by alkylating 1-[4-methoxyphenyl)-2-(4-(2-chloroethoxy)phenyl]-1-propanone under metal halogen exchange conditions with 1-iodo-6-methoxy-naphthalene. Following dehydration and cleavage of the methoxy groups, (E)-9b was formed by displacement of the chloro group with pyrrolidine. (E)-9b binding to ER generated calculated K(i) values of 3.7 nM for hER(alpha) and 72 nM for hER(beta). The antagonism of (E)-9b was demonstrated in cell transfection assays using the ERE from the vitA2 promotor and the natural ER-responsive pS2 promotor. With increasing concentrations of (E)-9b, the E(2)-dependent response was efficiently inhibited demonstrating that (E)-9b could function as an anti-estrogen in these assays. Interestingly, ER(alpha) activity was inhibited even below basal levels suggesting that ligand-independent activity of ER(alpha) was also inhibited. Computational docking studies suggest that the placement of the hydroxyl group on the naphthalene group may not be optimal and we are currently exploring additional analogues.     

De novo design of a monomeric three-stranded antiparallel beta-sheet

Here we describe the NMR conformational study of a 20-residue linear peptide designed to fold into a monomeric three-stranded antiparallel beta-sheet in aqueous solution. Experimental and statistical data on amino acid beta-turn and beta-sheet propensities, cross-strand side-chain interactions, solubility criteria, and our previous experience with beta-hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C-NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12-residue peptides encompassing the N- and C-segments of the designed sequence indicates that the 20-residue peptide folds into the expected conformation. Assuming a two-state model, the exchange kinetics between the beta-sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil-beta-sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in beta-sheet formation and stability.     

De novo design of a two-stranded coiled-coil switch peptide

The properties and characteristics shared by amyloid fibrils formed from disease and non-disease associated proteins that are unrelated in sequence and structure offer the prospect that model systems can be used to systematically assess the factors that predispose a native protein to form amyloid fibrils. Based on a de novo design approach, we recently reported a unique switch peptide model system, ccbeta, that forms a three-stranded coiled-coil structure at low temperatures and which can be easily converted to amyloid fibrils by increasing the temperature. To simplify the system further, we describe here the redesign of a two-stranded ccbeta coiled-coil variant and its detailed analysis by a variety of biophysical methods. Compared with the original design, the characteristics of the peptide make it even simpler to elucidate and validate fundamental principles of amyloid fibril-formation.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

De novo design of the hydrophobic cores of proteins.

We have developed and experimentally tested a novel computational approach for the de novo design of hydrophobic cores. A pair of computer programs has been written, the first of which creates a "custom" rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. The second program uses a genetic algorithm to globally optimize for a low energy core sequence and structure, using the custom rotamer library as input. Success of the programs in predicting the sequences of native proteins indicates that they should be effective tools for protein design. Using these programs, we have designed and engineered several variants of the phage 434 cro protein, containing five, seven, or eight sequence changes in the hydrophobic core. As controls, we have produced a variant consisting of a randomly generated core with six sequence changes but equal volume relative to the native core and a variant with a "minimalist" core containing predominantly leucine residues. Two of the designs, including one with eight core sequence changes, have thermal stabilities comparable to the native protein, whereas the third design and the minimalist protein are significantly destabilized. The randomly designed control is completely unfolded under equivalent conditions. These results suggest that rational de novo design of hydrophobic cores is feasible, and stress the importance of specific packing interactions for the stability of proteins. A surprising aspect of the results is that all of the variants display highly cooperative thermal denaturation curves and reasonably dispersed NMR spectra. This suggests that the non-core residues of a protein play a significant role in determining the uniqueness of the folded structure.     

De novo protein design using pairwise potentials and a genetic algorithm.

One of the major goals of molecular biology is to understand how protein chains fold into a unique 3-dimensional structure. Given this knowledge, perhaps the most exciting prospect will be the possibility of designing new proteins to perform designated tasks, an application that could prove to be of great importance in medicine and biotechnology. It is possible that effective protein design may be achieved without the requirement for a full understanding of the protein folding process. In this paper a simple method is described for designing an amino acid sequence to fit a given 3-dimensional structure. The compatibility of a designed sequence with a given fold is assessed by means of a set of statistically determined potentials (including interresidue pairwise and solvation terms), which have been previously applied to the problem of protein fold recognition. In order to generate sequences that best fit the fold, a genetic algorithm is used, whereby the sequence is optimized by a stochastic search in the style of natural selection.     

De novo design and synthesis of HIV-1 integrase inhibitors

Existing AIDS therapies are out of reach for most HIV-infected people in developing countries and, where available, they are limited by their toxicity and their cost. New anti-HIV agents are needed urgently to combat emerging viral resistance and reduce the side effects associated with currently available drugs. Toward this end, LeapFrog, a de novo drug design program was used to design novel, potent, and selective inhibitors of HIV-1 integrase. The designed compounds were synthesized and tested for in vitro inhibition of HIV-1 integrase. Out of the 25 compounds that were designed, and synthesized, four molecules (compounds 23, 26, 43, and 59) showed moderate to low inhibition of HIV-1 integrase for 3'-processing and 3'-strand transfer activities. Nonetheless, these compounds possess structural features not seen in known HIV-1 integrase inhibitors and thus can serve as excellent leads for further optimization of anti-HIV-1 integrase activity.     

Structure and dynamics of a helical hairpin and loop region in annexin 12: a site-directed spin labeling study

A 30-residue nitroxide scan encompassing a helical hairpin and an extended loop in soluble annexin 12 (helices D and E in repeat 2; residues 134-163) has been analyzed in terms of nitroxide side chain mobility and accessibility to collision with paramagnetic reagents (Pi). Values of Pi for both O(2) and a Ni(II) metal complex (NiEDDA) are remarkably well correlated with the fractional solvent accessibility of the native side chains at the corresponding positions computed from the known crystal structure. This result demonstrates the utility of Pi as an experimental measure of side chain accessibility in solution, as well as the lack of structural perturbation due to the presence of the nitroxide side chain. The pattern of side chain mobility is also in excellent agreement with predictions from the crystal structure. The results presented here extend the correlations between mobility and structure described in earlier work on other helical proteins, and suggest their generality. The periodic dependence of Pi and mobility along the sequence of annexin 12 reveals the helical segments and their orientation in the fold, as expected for a nonperturbing nitroxide side chain. However, these data do not distinguish the helix-loop-helix motif from a continuous helix, because immobilized side chains in the short loop sequence maintain the periodicity. As shown here, the ratio of Pi values for O(2) and NiEDDA clearly delineates the loop region, due to size exclusion effects between the two reagents. A new feature evident in a nitroxide scan through multiple secondary elements is a modulation of the basic Pi and mobility patterns along the sequence, apparently due to differences in helix packing and backbone motion. Thus, in the short helix D, residues are consistently more mobile and accessible throughout the sequence compared to the residues in the longer, less-solvated and more ordered helix E.     

De novo formation and ultra-structural characterization of a fiber-producing human hair follicle equivalent in vitro

Across many tissues and organs, the ability to create an organoid, the smallest functional unit of an organ, in vitro is the key both to tissue engineering and preclinical testing regimes. The hair follicle is an organoid that has been much studied based on its ability to grow quickly and to regenerate after trauma. But hair follicle formation in vitro has been elusive. Replacing hair lost due to pattern baldness or more severe alopecia, including that induced by chemotherapy, remains a significant unmet medical need.By carefully analyzing and recapitulating the growth conditions of hair follicle formation, we recreated human hair follicles in tissue culture that were capable of producing hair. Our microfollicles contained all relevant cell types and their structure and orientation resembled in some ways excised hair follicle specimens from human skin. This finding offers a new window onto hair follicle development. Having a robust culture system for hair follicles is an important step towards improved hair regeneration as well as to an understanding of how marketed drugs or drug candidates, including cancer chemotherapy, will affect this important organ.     

自组装ELP多肽的从头设计及其在生物医药领域中的应用进展

重组类弹性蛋白多肽(elastin-like polypeptides,ELPs)是一种通过基因工程方法合成的多肽聚合物,其结构由类弹性蛋白的肽段单元重复串连组成,具有刺激响应性、自组装特性、显著的弹性和良好的生物学特性,如低血小板黏附性和低免疫原性等,因此ELPs材料已被广泛应用于组织工程、药物输送和纳米生物器件制备...     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

旨在克隆、表达与纯化类弹性蛋白多肽,并测定类弹性蛋白的相变温度对不同的盐敏感程度。从头设计了类弹性蛋白多肽的序列并人工合成其编码基因片段,克隆至改造后的表达载体pET-22b中,构建重组表达载体,转化至大肠杆菌BL21(DE3) 中并诱导表达,采用可逆相变循环经高速离心对其进行纯化,并考察了盐类型及浓度对类弹性蛋白相变温度的影响。结果表明：0.4 mmol/L的Na2CO3能使25 μmol/L类弹性蛋白多肽 [KV8F-20] 相变温度降低至19 ℃,此类弹性蛋白多肽序列有望开发成一新型纯化标签,为今后     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker

Previously, we isolated and characterized attacin from Spodoptera exigua and a coleoptericin-like protein from Protaetia brevitarsis seulensis. In this study, we fused these two genes encoding antimicrobial proteins to obtain a hybrid protein with enhanced antimicrobial activity. To fuse the two antimicrobial proteins, we employed helical and non-helical linker sequences that function as inter-domain linkers in proteins. We used the Gly–Gly–Gly–Gly–Ser peptide as a non-helical linker. The hybrid protein produced using this linker showed less antimicrobial activity against Escherichia coli, Bacillus subtilis, Burkholderia glumae, Pseudomonas corrugate, and Erwinia rhapontici than either of the two parental antimicrobial proteins. In addition, the MIC value of the hybrid protein was 23.1 μM, which indicates poor activity against E. coli. When we used three Glu–Ala–Ala–Ala–Lys (EAAAK) peptide sequences as a helical linker to fuse the two proteins, the resultant hybrid protein had much higher antimicrobial activity than the parental antimicrobial proteins. In particular, this hybrid protein had strong antimicrobial activity against P. corrugate. These results indicate that the EAAAK motif can be used to effectively separate two antimicrobial proteins and produce a hybrid protein with more antimicrobial activity than either of the parent proteins.