Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation

Protein arrays will greatly accelerate research and development in medical and biological sciences. We have used cell-free protein biosynthesis and a parallel immobilization strategy for producing protein biochips. We demonstrate a model two-protein microarray using luciferase and green fluorescent protein, both expressed in a cell-free system and specifically immobilized on CombiMatrix semiconductor oligonucleotide microarrays. This demonstration provides evidence for the appropriate folding, activity, robust presentation, and efficient flexible detection of proteins on the microscale.     

Constraint-based assembly of tertiary protein structures from secondary structure elements

A challenge in computational protein folding is to assemble secondary structure elements-helices and strands-into well-packed tertiary structures. Particularly difficult is the formation of beta-sheets from strands, because they involve large conformational searches at the same time as precise packing and hydrogen bonding. Here we describe a method, called Geocore-2, that (1) grows chains one monomer or secondary structure at a time, then (2) disconnects the loops and performs a fast rigid-body docking step to achieve canonical packings, then (3) in the case of intrasheet strand packing, adjusts the side-chain rotamers; and finally (4) reattaches loops. Computational efficiency is enhanced by using a branch-and-bound search in which pruning rules aim to achieve a hydrophobic core and satisfactory hydrogen bonding patterns. We show that the pruning rules reduce computational time by 10(3)- to 10(5)-fold, and that this strategy is computationally practical at least for molecules up to about 100 amino acids long.     

Conjugational genetic exchange in the hyperthermophilic archaeon Sulfolobus acidocaldarius: intragenic recombination with minimal dependence on marker separation

In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.     

Synthesis and SAR of novel histamine H3 receptor antagonists

The synthesis and biological evaluation of novel tetrahydroisoquinoline, tetrahydroquinoline, and tetrahydroazepine antagonists of the human and rat H(3) receptors are described. The substitution around these rings as well as the nature of the substituent on nitrogen is explored. Several compounds with high affinity and selectivity for the human and rat H(3) receptors are reported.     

The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.     

Immunoassays based on electrochemical detection using microelectrode arrays

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.     

Cooperativity in two-state protein folding kinetics

We present a solvable model that predicts the folding kinetics of two-state proteins from their native structures. The model is based on conditional chain entropies. It assumes that folding processes are dominated by small-loop closure events that can be inferred from native structures. For CI2, the src SH3 domain, TNfn3, and protein L, the model reproduces two-state kinetics, and it predicts well the average Phi-values for secondary structures. The barrier to folding is the formation of predominantly local structures such as helices and hairpins, which are needed to bring nonlocal pairs of amino acids into contact.     

The worm re-turns: hiding behavior of a tube-dwelling marine polychaete, Serpula vermicularis

The polychaete worm Serpula vermicularis (Serpulidae) filterfeeds at the mouth of its calcareous tube, but retreats intothe tube when startled by mechanical stimuli likely to be associatedwith predators. While in its tube, a worm is safe but cannotfeed. Thus, hiding has a lost-opportunity cost. We show thatthis cost can be substantial, given that food in the naturalhabitat appears in pulses, and good feeding conditions may notlast long or recur frequently. We expect that a worm's hidingtime will be sensitive to the lost-opportunity cost, and wepresent data from a series of experiments that support thisprediction. The worms seem able to track relatively short-termchanges in food availability, and some evidence suggests thatthey assess food availability on a relative basis, comparingcurrent feeding conditions to those recently experienced. Hidingand other types of cryptic behavior are common antipredatortactics, and animals may commonly adjust the durations of suchbehaviors to current benefits and costs (including lost opportunity),as they perceive them     

A fast conformational search strategy for finding low energy structures of model proteins.

We describe a new computer algorithm for finding low-energy conformations of proteins. It is a chain-growth method that uses a heuristic bias function to help assemble a hydrophobic core. We call it the Core-directed chain Growth method (CG). We test the CG method on several well-known literature examples of HP lattice model proteins [in which proteins are modeled as sequences of hydrophobic (H) and polar (P) monomers], ranging from 20-64 monomers in two dimensions, and up to 88-mers in three dimensions. Previous nonexhaustive methods--Monte Carlo, a Genetic Algorithm, Hydrophobic Zippers, and Contact Interactions--have been tried on these same model sequences. CG is substantially better at finding the global optima, and avoiding local optima, and it does so in comparable or shorter times. CG finds the global minimum energy of the longest HP lattice model chain for which the global optimum is known, a 3D 88-mer that has only been reachable before by the CHCC complete search method. CG has the potential advantage that it should have nonexponential scaling with chain length. We believe this is a promising method for conformational searching in protein folding algorithms.