Higher Binding Rate Research Articles

Binding kinetics of lock and key colloids.

Using confocal microscopy and first passage time analysis, we measure and predict the rates of formation and breakage of polymer-depletion-induced bonds between lock-and-key colloidal particles and find that an indirect route to bond formation is accessed at a rate comparable to that of the direct formation of these bonds. In the indirect route, the pocket of the lock particle is accessed by nonspecific bonding of the key particle with the lock surface, followed by surface diffusion leading to specific binding in the pocket of the lock. The surprisingly high rate of indirect binding is facilitated by its high entropy relative to that of the pocket. Rate constants for forward and reverse transitions among free, nonspecific, and specific bonds are reported, compared to theoretical values, and used to determine the free energy difference between the nonspecific and specific binding states.

PePr: a peak-calling prioritization pipeline to identify consistent or differential peaks from replicated ChIP-Seq data.

ChIP-Seq is the standard method to identify genome-wide DNA-binding sites for transcription factors (TFs) and histone modifications. There is a growing need to analyze experiments with biological replicates, especially for epigenomic experiments where variation among biological samples can be substantial. However, tools that can perform group comparisons are currently lacking. We present a peak-calling prioritization pipeline (PePr) for identifying consistent or differential binding sites in ChIP-Seq experiments with biological replicates. PePr models read counts across the genome among biological samples with a negative binomial distribution and uses a local variance estimation method, ranking consistent or differential binding sites more favorably than sites with greater variability. We compared PePr with commonly used and recently proposed approaches on eight TF datasets and show that PePr uniquely identifies consistent regions with enriched read counts, high motif occurrence rate and known characteristics of TF binding based on visual inspection. For histone modification data with broadly enriched regions, PePr identified differential regions that are consistent within groups and outperformed other methods in scaling False Discovery Rate (FDR) analysis. http://code.google.com/p/pepr-chip-seq/.

ABSTRACT 795

Background and aims: Colchicine poisoning is an uncommon, but potentially life-threatening, toxicologic emergency. It has a narrow therapeutic index and even death can be occur in minor toxicity. Initial 36 to 72 hours after ingestion, death might be seen caused by hypovolemic shock, central nervous system damage, and cardiopulmonary insufficiency. All patients who had colchicine overdose should be followed and treated in the intensive care units and treatment is usually supportive. Aims: In this case, we presented a patient diagnosed as colchicine intoxication and treated with plasma exchange. Methods: A previously healthy 6-year-old girl was ingested lethal doses of colchicium and referred to our pediatric intensive care unit (PICU). During the admission she had acute respiratory distress syndrome (ARDS), elevated transaminase levels, diarrhea and coagulopathy. The two plasma exchange session were applied to the patient who had multiorgan insufficiency already. Results: Drug removal during plasmapheresis are sparse, -mostly case reports of overdoses have described the effects of plasmapheresis on pharmaceutical. In general, drugs with a low volume of distribution (Vd) and/or a high rate of protein binding are most likely to be removed during plasma exchange. Colchicine completely does not have these features. But in some cases, plasma exchange might be beneficial in the treatment of colchicine intoxications. Conclusions: As a result, we consider that plasma exchange can be beneficial in eradication of colchicine from the body as it tightly binds to the plasma proteins and also decrease the mortality caused by its intoxication.

Ammonia Leaching Study on Refractory Oxidised Copper Ores

Ammonia leaching research is carried out on a certain oxidised copper ores. The effects of oxidized copper ores which came from Yunnan Province were studied in laboratory in order to find out the optimum leaching conditions under normal temperature and pressure. The ores belonged to typical high binding rate minerals. The optimum conditions obtained were as follows: granding fineness about 90% -0.074mm, ammonia concentration 2 mol/l, ammonium fluoride concentration 1.5mol/l , leaching time 2h, and liquid-solid ratio 3:1.The leaching rate of this ore could reach 86%.

Spectral characteristics of the mutant form GGBP/H152C of D-glucose/D-galactose-binding protein labeled with fluorescent dye BADAN: influence of external factors

The mutant form GGBP/H152C of the D-glucose/D-galactose-binding protein with the solvatochromic dye BADAN linked to cysteine residue Cys 152 can be used as a potential base for a sensitive element of glucose biosensor system. We investigated the influence of various external factors on the physical-chemical properties of GGBP/H152C-BADAN and its complex with glucose. The high affinity (Kd = 8.5 µM) and high binding rate of glucose make GGBP/H152C-BADAN a good candidate to determine the sugar content in biological fluids extracted using transdermal techniques. It was shown that changes in the ionic strength and pH of solution within the physiological range did not have a significant influence on the fluorescent characteristics of GGBP/H152C-BADAN. The mutant form GGBP/H152C has relatively low resistance to denaturation action of GdnHCl and urea. This result emphasizes the need to find more stable proteins for the creation of a sensitive element for a glucose biosensor system.

Inhibitory and Fungicidal Effects of Antifungal Drugs against Aspergillus Species in the Presence of Serum

Given the high protein binding rates of antifungal drugs and the effect of serum proteins on Aspergillus growth, we investigated the in vitro pharmacodynamics of amphotericin B, voriconazole, and three echinocandins in the presence of human serum, assessing both inhibitory and fungicidal effects. In vitro inhibitory (IC) and fungicidal (FC) concentrations against 5 isolates of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus were determined with a CLSI M38-A2-based microdilution method using the XTT methodology after 48 h of incubation at 35 °C with a medium supplemented with 50% human serum. In the presence of serum, the IC and FC of amphotericin B and the IC of echinocandins were increased (1.21- to 13.44-fold), whereas voriconazole IC and FC were decreased (0.22- to 0.90-fold). The amphotericin B and voriconazole FC/IC ratios did not change significantly (0.59- to 2.33-fold) in the presence of serum, indicating that the FC increase was due to the IC increase. At echinocandin concentrations above the minimum effective concentration (MEC), fungal growth was reduced by 10 to 50% in the presence of human serum, resulting in complete inhibition of growth for some isolates. Thus, the in vitro activities of amphotericin B and echinocandins were reduced, whereas that of voriconazole was enhanced, in the presence of serum. These changes could not be predicted by the percentage of protein binding, indicating that other factors and/or secondary mechanisms may account for the observed in vitro activities of antifungal drugs against Aspergillus species in the presence of serum.

A case of insulin antibody-induced glucose instability in an elderly woman with type 2 diabetes on hemodialysis, successfully ameliorated with liraglutide

We encountered a 68-year-old woman with type 2 diabetes who had frequent insulin-induced hypoglycemia in the morning. She had renal failure, and was on hemodialysis. The hypoglycemia in the morning was severe, induced by the small dosage of insulin, whilst she showed severe postprandial hyperglycemia. A blood test revealed high serum insulin and C-peptide levels, and a high binding rate of insulin antibody. Switching human insulin to an insulin analogue, glulisine, failed to avoid hypoglycemia. Scatchard plot analysis indicated that the high affinity site of the patient’s insulin antibody had a low affinity constant (K1) and a high binding capacity (R1) against all tested insulin, almost equivalent values reported in insulin autoimmune syndrome cases. Treatment with the glucagon-like peptide-1 analogue liraglutide ameliorated the postprandial hyperglycemia without hypoglycemia, resulting in improved glycemic stability. Furthermore, the continued use of liraglutide reduced both serum insulin level and binding rate of insulin antibody. This is the first case of using liraglutide in an insulin-antibody positive patient with renal failure on hemodialysis. Liraglutide could be an effective and safe remedy for patients with insulin antibody-related glucose instability, regardless of their renal function.

Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

Identifying the Molecular Mechanics and Binding Dynamics Characteristics of Potent Inhibitors to HIV‐1 Protease

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the primary inhibition targets for chemotherapy of AIDS because of its critical role in the replication cycle of the HIV. In this work, a combinatory coarse-grained and atomistic simulation method was developed for dissecting molecular mechanisms and binding process of inhibitors to the active site of HIV-1 PR, in which 35 typical inhibitors were trialed. We found that the molecular size and stiffness of the inhibitors and the binding energy between the inhibitors and PR play important roles in regulating the binding process. Comparatively, the smaller and more flexible inhibitors have larger binding energy and higher binding rates; they even bind into PR without opening the flaps. In contrast, the larger and stiffer inhibitors have lower binding energy and lower binding rate, and their binding is subjected to the opening and gating of the PR flaps. Furthermore, the components of binding free energy were quantified and analyzed by their dependence on the molecular size, structures, and hydrogen bond networks of inhibitors. Our results also deduce significant dynamics descriptors for determining the quantitative structure and property relationship in potent drug ligands for HIV-1 PR inhibition.

Abstract 510: Identification and characterization of immunogenic epitopes from CD133 and their potential for use to immunologically target cancer stem cells

Abstract CD133 is a marker that identifies cancer stem cells (CSC) on many solid tumors and its expression has been correlated with shortened survival. Although expressed on many normal tissues and stem cells, CD133 is overexpressed on CSC and normal SC are negative or express low levels of MHC making them less sensitive to recognition by CD8+ cytotoxic T lymphocytes (CTL). Potential CTL epitopes were identified by algorithms to predict binding to HLA-A2. Binding studies showed high affinity binding and low off rates for four candidate epitopes. CTL induction studies using IL-12 secreting dendritic cells to stimulate CD8+ T cells evaluated the immunogenicity of the candidate peptides after three or four weekly in vitro stimulations. Multimer and IFNγ responses were observed with CD133-753, 405 and 708 epitopes. Subsequent studies demonstrated killing of T2 cells loaded with peptide. To evaluate the potential for autoimmunity, mouse homolog peptides of the 753 and 405 epitopes that were shown to have high affinity binding to human HLA-A2 were used to immunize HLA-A2 transgenic mice (9CB6F1-Tg (HLA-A*0201/H2-Kb) A*0201). Mice were immunized 3 times at 3 week intervals in IFA + HBV helper peptide and spleens were harvested 2 weeks after the last immunization and stimulated in vitro for one week with peptide pulsed LPS stimulated- APC. An additional group was sacrificed 6 weeks after the last immunization and studied. IFNγ ELISpot assays showed immune responses to the 753 and 405 peptides in 35% and 40% of mice. Organs, including heart, lung, liver, kidney, stomach, intestine, brain, bone marrow, gonads, and eyes from mice with immune responses were found to be negative for lymphocytic infiltrations supporting a lack of autoimmunity related to the immune response to these peptides. Together these studies support the safety and immunogenicity of these peptides as a potential vaccine to target CD133 cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 510. doi:1538-7445.AM2012-510

Abstract 3792: Population Pharmacokinetic (PPK) modeling of ProLindac™ (AP5346), a nanopolymer prodrug of diaminocyclohexane (DACH)-platinum (Pt) in recurrent advanced ovarian cancer (AOC) patients

Abstract Background: ProLindac™ (PL) is a 25 kDa hydroxypropylmethacrylamide copolymer linked to a DACH-Pt. The DACH-Pt complex is thought to be biologically inert while attached to the polymer, with slow in vivo Pt release accelerated in low pH environments (extracellular space of hypoxic tumor and intracellular lysosomal compartment). Methods: 9/35 patients (pts) with recurrent OAC treated with single-agent PL via 2-h IV infusion at doses of 900 mg/m*2 q2w (3 pts), 1400 mg/m*2 q3w (3 pts) and 1680 mg/m*2 q3w (3 pts) were assessable for full PK analysis. 8 time point samples were collected on cycle 1, and 2 samples at cycles 2, 3 and 4. The ultrafiltration procedure used Ultra-4 filters (Amicon) with 50 kDa for unbound PL (U-PL) and 3 kDa for unbound Pt (U-Pt) cut-off. Urine was collected every 6 h from day 1 to 5 of dosing 1. Pt levels were measured by a validated Flameless Atomic Absorption Spectrometry Method (concentration range: 12.5 - 500 ng/mL). PPK was modeled using a nonlinear mixed effect model program (Monolix version 3.1s) by computing the maximum likelihood estimation of the parameters without any approximation of the model. Results: A 3-compartment open model adequately described the total PL time-concentration curve with linear elimination while a 2-compartment model satisfactorily described U-PL and U-Pt time-concentration curves with linear elimination. Mean PPK parameters with SD are summarized below: The mean recovery of PL dose in urine was at least 60% within 5 days. Conclusion: The central Vd for total PL Pt is rather small, close to the circulating blood volume suggesting that the major part of parent drug is restrained in the circulatory space with weak tissue diffusion probably due to a high protein binding rate. However, for U-PL and U-Pt, the Vd is much higher suggesting: 1) a significant tissue diffusion of active species (free-Pt), 2) PL is bound to plasma proteins with low-energy bonds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3792. doi:1538-7445.AM2012-3792

Ligation tunes protein reactivity in an ancient haemoglobin: kinetic evidence for an allosteric mechanism in Methanosarcina acetivorans protoglobin.

Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin with peculiar structural properties such as a completely buried haem and two orthogonal tunnels connecting the distal cavity to the solvent. CO binding to and dissociation from MaPgb occur through a biphasic kinetics. We show that the heterogenous kinetics arises from binding to (and dissociation from) two tertiary conformations in ligation-dependent equilibrium. Ligation favours the species with high binding rate (and low dissociation rate). The equilibrium is shifted towards the species with low binding (and high dissociation) rates for the unliganded molecules. A quantitative model is proposed to describe the observed carbonylation kinetics.

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

Author SummaryNoncovalent biological interactions are commonly subjected to mechanical force, particularly when they are involved in adhesion or cytoskeletal movements. While one might expect mechanical force to break these interactions, some of them form so-called catch bonds that lock on harder under force, like a nanoscale finger-trap. In this study, we show that the catch-bond forming adhesive protein FimH, which is located at the tip of E. coli fimbriae, allows bacteria to bind to urinary epithelial cells in a shear-dependent manner; that is, they bind at high but not at low flow. We show that isolated fimbrial tips, consisting of elongated protein complexes with FimH at the apex, reproduce this behavior in vitro. Our molecular dynamics simulations of the fimbrial tip structure show that FimH is shaped like a hook that is normally rigid but opens under force, causing structural changes that lead to firm anchoring of the bacteria on the surface. In contrast, the more distal adaptor proteins of the fimbrial tip create a flexible connection of FimH to the rigid fimbria, enhancing the ability of the adhesin to move into position and form bonds with mannose on the surface. We suggest that the entire tip complex forms a hook-chain, ideal for rapid and stable anchoring in flow.

Evaluating the activity of the RNA polymerase inhibitor myxopyronin B against Staphylococcus aureus

Myxopyronin B (MyxB) binds to the switch region of RNA polymerase (RNAP) and inhibits transcriptional initiation. To evaluate the potential development of MyxB as a novel class of antibiotic, we characterized the antimicrobial activity of MyxB against Staphylococcus aureus. Spontaneous MyxB resistance in S. aureus occurred at a frequency of 8 × 10(-8) , similar to that of rifampin. The MyxB-resistant mutants were found to be altered in single amino acid residues in the RNAP subunits that form the MyxB-binding site. In the presence of human serum albumin, the MyxB minimum inhibitory concentration against S. aureus increased drastically (≥128-fold) and 99.5% of MyxB was protein bound. Because of the high serum protein binding and resistance rate, we conclude that MyxB is not a viable starting point for antibiotic development.

PRODUCTION OF ANTI-PMSG ANTIBODIES AND ITS RELATION TO THE PRODUCTIVITY OF RABBIT DOES

A batch of 100 female rabbits of the Hyplus breed were subjected to artificial insemination (A.I.) for a period of 9 months. With the goal of inducing the sexual receptivity of the does, half of them received systematic doses of 25 I.U .. of PMSG (Ciclogonine PROCHENA), 48 hours before A.I.. The other half were not injected (Control group). Following this experimental period (40 days after the last injection), blood samples of all the does were taken, in order to assay the anti-PMSG antibodies and to study the relationship between the production of antibodies, and the receptivity and productivity of the does observad for the previous litter. For the PMSG group, we classified the results of the does according to their level of immunity which either has a low binding rate (≤4 %) or a high one (> 4 %). 30,6% of the does from the group treated with PMSG produced anti­PMSG antibodies with a high binding rate. In no way related to the level of production of the antibodies, the PMSG treated does were found to be significantly more receptiva than the does in the control group (respectively 70.6 - 66.7 vs 42.0%, P = 0.023), while the fertility did not appear to have been either improved or decreased by the PMSG treatment. The total number of young born, born alive and weaned rabbits per litter did not vary according to the treatment or the intensity of antibody production. Likewise the weight of the litter and also the individual mean weight of the weaned young did not vary significantly according to the group. However, inside of the group treated with PMSG, the individual weight at weaning was the highest for the young from hyper- immune mothers (710 vs 639g, P = 0.035). For 29 does from the PMSG group, a second blood sample was taken 12 weeks after the previous one. Only 3 does showed a decreased level of antibodies, 4 months after the last injection. The study showed that one third of the does developed an immune reaction following injections of PMSG; however the proportion of antibodies produced did not appear related to either their sexual receptivity or productivity.

LC-ESI-MS-MS Determination of Rat Plasma Protein Binding of Major Flavonoids of Flos Lonicerae Japonicae by Centrifugal Ultrafiltration

Centrifugal ultrafiltration (CU) and a fast liquid chromatography with electrospray ionization tandem mass spectrometric (LC-ESI-MS-MS) method have been developed for drug-protein binding analysis of four flavonoids of Flos Lonicerae Japonicae (FLJ). A rapid separation of free drug from the plasma was achieved by CU and the simultaneous analysis of four flavonoids of FLJ in rat plasma was by fast LC-ESI-MS-MS. The chromatographic analytical time decreased to 6.5 min without sacrificing resolution using a 4.6 mm × 50 mm, 1.8 μm particle size reverse phase column and MS–MS mode performed for multiple reaction monitoring (MRM). The protein binding rates of four flavonoids were in the range of 66.8 ± 7.4 to 81.3 ± 5.1% after oral administration of the flavonoid fraction of FLJ. All values were almost similar to 70.0% at 30, 45 and 80 min. Its result suggests that these flavonoids in rat plasma perform high protein binding rates and present a stable binding in a longer time. Meanwhile, it reveals that the elimination of these flavonoids may be relatively moderate in the body, the performance of a certain degree of accumulation may be speculated and make further impacts on the pharmacokinetic parameters.

Direct Activators BID & BIM Function Like Membrane Receptors for BAX & BCL-XL

The BCL-2 protein family is a primary regulator of apoptosis and its interaction network converges at the pro-apoptotic BAX/BAK nexus. BAX has soluble, membrane-bound, and membrane-integrated forms that are central to the management of mitochondrial permeabilization. These states, which lead to BAX pore formation and cytochrome c egress, are modulated by anti-apoptotic multidomain and pro-apoptotic BH3-only proteins. Using purified recombinant BCL-2 proteins and defined liposomes, the soluble->membrane transitions and pore activity modulations have been characterized.Direct activators cBID and BIMS instigate BAX pore formation - a process inhibitable by BCL-XL - and these oppositional functions are dosage-dependent. BIMS is more efficient an activator than cBID; however, BIMS-BAX activation is more susceptible to inhibition by BCL-XL. Since the steps of BAX activation remain controversial, we investigated the kinetics of protein-membrane binding. BAX, cBID, BIMS, and BCL-XL are each capable of adsorbing to membranes, albeit with differing properties. These proteins' transitions to lipid bilayers include a rapid binding step that is reversible and distinct from a slower membrane integration step. BCL-XL and BIMS show a comparatively high rate of binding to membranes whereas BAX and cBID are substantially slower. The membrane-resident forms of each protein have comparably strong affinities for membranes indicating that the on-rate is most influential on their in-membrane concentrations.The difference in membrane on-rates between the direct activators potentially accounts for the disparity in their BAX activation efficacies. Intriguingly, the membrane-resident forms of cBID and BIMS were capable of driving BAX and BCL-XL to tight membrane affinity conformations. These activities were saturable, suggesting a protein-protein interaction rather than modulation of the bulk membrane environment. These data reveal receptor-like roles for cBID & BIMS for soluble BCL-2 proteins during the initiation of apoptosis.

Binding Rate Screen – A high-throughput assay in soluble lysate for prioritizing protein expression constructs

Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His 6) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10–100 times faster than any other technologies currently available.

UV cross-linking of unmodified DNA on glass surfaces

The performance of DNA microarrays strongly depends on their surface properties. Furthermore, the immobilization method of the capture molecules is of importance for the efficiency of the microarray in terms of sensitivity and specificity. This work describes the immobilization of single-stranded capture oligonucleotides by UV cross-linking on silanated (amino and epoxy) glass surfaces. Thereby we used amino (NH2) and poly thymine/poly cytosine modifications of the capture sequences as well as unmodified capture molecules. The results were compared to UV cross-linking of the same DNA oligonucleotides on unmodified glass surfaces. Immobilization and hybridization efficiency was demonstrated by fluorescence and enzyme-induced deposition of silver nanoparticles. We found out that single-stranded DNA molecules do not require a special modification to immobilize them by UV cross-linking on epoxy- or amino-modified glass surfaces. However, higher binding rates can be achieved when using amino-modified oligonucleotides on an epoxy surface. The limit of detection for the used settings was 5 pM.

Dissecting the High Rate Constant for the Binding of a Ribotoxin to the Ribosome

Restrictocin belongs to a family of site-specific ribonucleases that kill cells by inactivating the ribosome. The restrictocin-ribosome binding rate constant was observed to exceed 1010 M−1s−1 [1]. We have developed a transient-complex theory to model the binding rates of protein-protein and protein-RNA complexes [2, 3]. The theory predicts the rate constant as ka = ka0 exp(-Uel∗/kBT), where ka0 is the basal rate constant for reaching the transient complex, located at the outer boundary of the bound state, by random diffusion, and Uel∗ is the average electrostatic interaction energy within the transient complex. Here we applied the transient-complex theory to dissect the high restrictocin-ribosome binding rate constant. We found that the binding rate of restrictocin to the isolated sarcin/ricin loop is electrostatically enhanced by ∼300-fold, similar to results found in protein-protein and protein-RNA complexes [2, 3]. Ribosome provides an additional 5000-fold rate enhancement. Two mechanisms work together to provide the dramatic additional enhancement. First, with the ribosome the transient complex is formed with relative separations and orientations where local electrostatic interactions with sarcin/ricin loop are particularly favorable. Second, distant parts of the ribosome provides additional electrostatic attraction with the ribotoxin. Our results quantitatively rationalize the experimental results for salt dependences and mutational effects of the binding rates of restrictocin with the isolated sarcin/ricin loop and the ribosome. [1] Korennykh, A. V., Piccirilli, J. A., and Correll, C. C. (2006) Nat. Struct. Mol. Biol. 13, 436-443. [2] Alsallaq, R. and Zhou, H.-X. (2007). Structure 15, 215-224. [3] Qin, S.B. and Zhou, H.-X. (2008). J. Phys. Chem. B 112, 5955-5960.