skip to main content


Title: Aquaglyceroporin AQP7's affinity for its substrate glycerol. Have we reached convergence in the computed values of glycerol-aquaglyceroporin affinity?
AQP7 is one of the four human aquaglyceroporins that facilitate glycerol transport across the cell membrane, a biophysical process that is essential in human physiology. Therefore, it is interesting to compute AQP7's affinity for its substrate (glycerol) with reasonable certainty to compare with the experimental data suggesting high affinity in contrast with most computational studies predicting low affinity. In this study aimed at computing the AQP7-glycerol affinity with high confidence, we implemented a direct computation of the affinity from unbiased equilibrium molecular dynamics (MD) simulations of three all-atom systems constituted with 0.16 M, 4.32 M, and 10.23 M atoms, respectively. These three sets of simulations manifested a fundamental physics law that the intrinsic fluctuations of pressure in a system are inversely proportional to the system size (the number of atoms in it). These simulations showed that the computed values of glycerol-AQP7 affinity are dependent upon the system size (the inverse affinity estimations were, respectively, 47.3 mM, 1.6 mM, and 0.92 mM for the three model systems). In this, we obtained a lower bound for the AQP7-glycerol affinity (an upper bound for the dissociation constant). Namely, the AQP7-glycerol affinity is stronger than 1087/M (the dissociation constant is less than 0.92 mM). Additionally, we conducted hyper steered MD (hSMD) simulations to map out the Gibbs free-energy profile. From the free-energy profile, we produced an independent computation of the AQP7-glycerol dissociation constant being approximately 0.18 mM.  more » « less
Award ID(s):
2031576
NSF-PAR ID:
10427401
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
RSC Advances
Volume:
12
Issue:
5
ISSN:
2046-2069
Page Range / eLocation ID:
3128 to 3135
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Dibenzo-18-crown-6 (DB18C6) is a single-crown ether that can act as a host for a guest ion. In an effort to illuminate the relationships among structure, dynamics, and thermodynamics of ligand binding in a simple model for understanding the affinity and specificity of ligand interactions, nuclear magnetic resonance (NMR) experiments and density functional theory (DFT) were used to study the interaction of DB18C6 with ammonium ion. 1H-NMR was used to follow the titration of DB18C6 with ammonium chloride in deuterated methanol, a solvent chosen for its amphipathic character. Ammonium ion binds strongly to DB18C6 with a dissociation equilibrium constant at least as low as ~ 10 - 6 M. DFT calculations were used to identify optimized conformations of bound and free DB18C6 and to estimate its binding energy with ammonium ion in implicit solvent. An approach is described that accounts for geometry relaxation in addition to solvation correction and basis set superposition error; to our knowledge, this is the first such report that includes the energy difference from optimizing species geometry. The lowest-energy conformer of free DB18C6 in implicit methanol acquires an open, W-shaped structure that is also the lowest-energy conformer found for the DB18C6-ammonium ion complex. These results form a foundation for further studies of this system by molecular dynamics simulations. 
    more » « less
  2. Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody‐binding protein as an adapter can simplify antibody functionalization by forming a specific antibody‐bound complex and introducing site‐specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody‐binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein (“lasso”), which binds human immunoglobulin G1 (IgG1) withKD = 0.53 nmand a dissociation rate that is 55‐ to 84‐fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme‐linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12‐fold. The small size of the lasso and a long half‐life of dissociation make the peptide a useful tool in antibody detection and immobilization.

     
    more » « less
  3. Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20-0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents. 
    more » « less
  4. Site description. This data package consists of data obtained from sampling surface soil (the 0-7.6 cm depth profile) in black mangrove (Avicennia germinans) dominated forest and black needlerush (Juncus roemerianus) saltmarsh along the Gulf of Mexico coastline in peninsular west-central Florida, USA. This location has a subtropical climate with mean daily temperatures ranging from 15.4 °C in January to 27.8 °C in August, and annual precipitation of 1336 mm. Precipitation falls as rain primarily between June and September. Tides are semi-diurnal, with 0.57 m median amplitudes during the year preceding sampling (U.S. NOAA National Ocean Service, Clearwater Beach, Florida, station 8726724). Sea-level rise is 4.0 ± 0.6 mm per year (1973-2020 trend, mean ± 95 % confidence interval, NOAA NOS Clearwater Beach station). The A. germinans mangrove zone is either adjacent to water or fringed on the seaward side by a narrow band of red mangrove (Rhizophora mangle). A near-monoculture of J. roemerianus is often adjacent to and immediately landward of the A. germinans zone. The transition from the mangrove to the J. roemerianus zone is variable in our study area. An abrupt edge between closed-canopy mangrove and J. roemerianus monoculture may extend for up to several hundred meters in some locations, while other stretches of ecotone present a gradual transition where smaller, widely spaced trees are interspersed into the herbaceous marsh. Juncus roemerianus then extends landward to a high marsh patchwork of succulent halophytes (including Salicornia bigellovi, Sesuvium sp., and Batis maritima), scattered dwarf mangrove, and salt pans, followed in turn by upland vegetation that includes Pinus sp. and Serenoa repens. Field design and sample collection. We established three study sites spaced at approximately 5 km intervals along the western coastline of the central Florida peninsula. The sites consisted of the Salt Springs (28.3298°, -82.7274°), Energy Marine Center (28.2903°, -82.7278°), and Green Key (28.2530°, -82.7496°) sites on the Gulf of Mexico coastline in Pasco County, Florida, USA. At each site, we established three plot pairs, each consisting of one saltmarsh plot and one mangrove plot. Plots were 50 m^2 in size. Plots pairs within a site were separated by 230-1070 m, and the mangrove and saltmarsh plots composing a pair were 70-170 m apart. All plot pairs consisted of directly adjacent patches of mangrove forest and J. roemerianus saltmarsh, with the mangrove forests exhibiting a closed canopy and a tree architecture (height 4-6 m, crown width 1.5-3 m). Mangrove plots were located at approximately the midpoint between the seaward edge (water-mangrove interface) and landward edge (mangrove-marsh interface) of the mangrove zone. Saltmarsh plots were located 20-25 m away from any mangrove trees and into the J. roemerianus zone (i.e., landward from the mangrove-marsh interface). Plot pairs were coarsely similar in geomorphic setting, as all were located on the Gulf of Mexico coastline, rather than within major sheltering formations like Tampa Bay, and all plot pairs fit the tide-dominated domain of the Woodroffe classification (Woodroffe, 2002, "Coasts: Form, Process and Evolution", Cambridge University Press), given their conspicuous semi-diurnal tides. There was nevertheless some geomorphic variation, as some plot pairs were directly open to the Gulf of Mexico while others sat behind keys and spits or along small tidal creeks. Our use of a plot-pair approach is intended to control for this geomorphic variation. Plot center elevations (cm above mean sea level, NAVD 88) were estimated by overlaying the plot locations determined with a global positioning system (Garmin GPS 60, Olathe, KS, USA) on a LiDAR-derived bare-earth digital elevation model (Dewberry, Inc., 2019). The digital elevation model had a vertical accuracy of ± 10 cm (95 % CI) and a horizontal accuracy of ± 116 cm (95 % CI). Soil samples were collected via coring at low tide in June 2011. From each plot, we collected a composite soil sample consisting of three discrete 5.1 cm diameter soil cores taken at equidistant points to 7.6 cm depth. Cores were taken by tapping a sleeve into the soil until its top was flush with the soil surface, sliding a hand under the core, and lifting it up. Cores were then capped and transferred on ice to our laboratory at the University of South Florida (Tampa, Florida, USA), where they were combined in plastic zipper bags, and homogenized by hand into plot-level composite samples on the day they were collected. A damp soil subsample was immediately taken from each composite sample to initiate 1 y incubations for determination of active C and N (see below). The remainder of each composite sample was then placed in a drying oven (60 °C) for 1 week with frequent mixing of the soil to prevent aggregation and liberate water. Organic wetland soils are sometimes dried at 70 °C, however high drying temperatures can volatilize non-water liquids and oxidize and decompose organic matter, so 50 °C is also a common drying temperature for organic soils (Gardner 1986, "Methods of Soil Analysis: Part 1", Soil Science Society of America); we accordingly chose 60 °C as a compromise between sufficient water removal and avoidance of non-water mass loss. Bulk density was determined as soil dry mass per core volume (adding back the dry mass equivalent of the damp subsample removed prior to drying). Dried subsamples were obtained for determination of soil organic matter (SOM), mineral texture composition, and extractable and total carbon (C) and nitrogen (N) within the following week. Sample analyses. A dried subsample was apportioned from each composite sample to determine SOM as mass loss on ignition at 550 °C for 4 h. After organic matter was removed from soil via ignition, mineral particle size composition was determined using a combination of wet sieving and density separation in 49 mM (3 %) sodium hexametaphosphate ((NaPO_3)_6) following procedures in Kettler et al. (2001, Soil Science Society of America Journal 65, 849-852). The percentage of dry soil mass composed of silt and clay particles (hereafter, fines) was calculated as the mass lost from dispersed mineral soil after sieving (0.053 mm mesh sieve). Fines could have been slightly underestimated if any clay particles were burned off during the preceding ignition of soil. An additional subsample was taken from each composite sample to determine extractable N and organic C concentrations via 0.5 M potassium sulfate (K_2SO_4) extractions. We combined soil and extractant (ratio of 1 g dry soil:5 mL extractant) in plastic bottles, reciprocally shook the slurry for 1 h at 120 rpm, and then gravity filtered it through Fisher G6 (1.6 μm pore size) glass fiber filters, followed by colorimetric detection of nitrite (NO_2^-) + nitrate (NO_3^-) and ammonium (NH_4^+) in the filtrate (Hood Nowotny et al., 2010,Soil Science Society of America Journal 74, 1018-1027) using a microplate spectrophotometer (Biotek Epoch, Winooski, VT, USA). Filtrate was also analyzed for dissolved organic C (referred to hereafter as extractable organic C) and total dissolved N via combustion and oxidation followed by detection of the evolved CO_2 and N oxide gases on a Formacs HT TOC/TN analyzer (Skalar, Breda, The Netherlands). Extractable organic N was then computed as total dissolved N in filtrate minus extractable mineral N (itself the sum of extractable NH_4-N and NO_2-N + NO_3-N). We determined soil total C and N from dried, milled subsamples subjected to elemental analysis (ECS 4010, Costech, Inc., Valencia, CA, USA) at the University of South Florida Stable Isotope Laboratory. Median concentration of inorganic C in unvegetated surface soil at our sites is 0.5 % of soil mass (Anderson, 2019, Univ. of South Florida M.S. thesis via methods in Wang et al., 2011, Environmental Monitoring and Assessment 174, 241-257). Inorganic C concentrations are likely even lower in our samples from under vegetation, where organic matter would dilute the contribution of inorganic C to soil mass. Nevertheless, the presence of a small inorganic C pool in our soils may be counted in the total C values we report. Extractable organic C is necessarily of organic C origin given the method (sparging with HCl) used in detection. Active C and N represent the fractions of organic C and N that are mineralizable by soil microorganisms under aerobic conditions in long-term soil incubations. To quantify active C and N, 60 g of field-moist soil were apportioned from each composite sample, placed in a filtration apparatus, and incubated in the dark at 25 °C and field capacity moisture for 365 d (as in Lewis et al., 2014, Ecosphere 5, art59). Moisture levels were maintained by frequently weighing incubated soil and wetting them up to target mass. Daily CO_2 flux was quantified on 29 occasions at 0.5-3 week intervals during the incubation period (with shorter intervals earlier in the incubation), and these per day flux rates were integrated over the 365 d period to compute an estimate of active C. Observations of per day flux were made by sealing samples overnight in airtight chambers fitted with septa and quantifying headspace CO_2 accumulation by injecting headspace samples (obtained through the septa via needle and syringe) into an infrared gas analyzer (PP Systems EGM 4, Amesbury, MA, USA). To estimate active N, each incubated sample was leached with a C and N free, 35 psu solution containing micronutrients (Nadelhoffer, 1990, Soil Science Society of America Journal 54, 411-415) on 19 occasions at increasing 1-6 week intervals during the 365 d incubation, and then extracted in 0.5 M K_2SO_4 at the end of the incubation in order to remove any residual mineral N. Active N was then quantified as the total mass of mineral N leached and extracted. Mineral N in leached and extracted solutions was detected as NH_4-N and NO_2-N + NO_3-N via colorimetry as above. This incubation technique precludes new C and N inputs and persistently leaches mineral N, forcing microorganisms to meet demand by mineralizing existing pools, and thereby directly assays the potential activity of soil organic C and N pools present at the time of soil sampling. Because this analysis commences with disrupting soil physical structure, it is biased toward higher estimates of active fractions. Calculations. Non-mobile C and N fractions were computed as total C and N concentrations minus the extractable and active fractions of each element. This data package reports surface-soil constituents (moisture, fines, SOM, and C and N pools and fractions) in both gravimetric units (mass constituent / mass soil) and areal units (mass constituent / soil surface area integrated through 7.6 cm soil depth, the depth of sampling). Areal concentrations were computed as X × D × 7.6, where X is the gravimetric concentration of a soil constituent, D is soil bulk density (g dry soil / cm^3), and 7.6 is the sampling depth in cm. 
    more » « less
  5. Molecular dynamics (MD) simulations generate valuable all-atom resolution trajectories of complex systems, but analyzing this high-dimensional data as well as reaching practical timescales, even with powerful supercomputers, remain open problems. As such, many specialized sampling and reaction coordinate construction methods exist that alleviate these problems. However, these methods typically don't work directly on all atomic coordinates, and still require previous knowledge of the important distinguishing features of the system, known as order parameters (OPs). Here we present AMINO, an automated method that generates such OPs by screening through a very large dictionary of OPs, such as all heavy atom contacts in a biomolecule. AMINO uses ideas from information theory to learn OPs that can then serve as an input for designing a reaction coordinate which can then be used in many enhanced sampling methods. Here we outline its key theoretical underpinnings, and apply it to systems of increasing complexity. Our applications include a problem of tremendous pharmaceutical and engineering relevance, namely, calculating the binding affinity of a protein–ligand system when all that is known is the structure of the bound system. Our calculations are performed in a human-free fashion, obtaining very accurate results compared to long unbiased MD simulations on the Anton supercomputer, but in orders of magnitude less computer time. We thus expect AMINO to be useful for the calculation of thermodynamics and kinetics in the study of diverse molecular systems. 
    more » « less