Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases.

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme-reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament.

The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158-166) and a highly flexible region (residues 167-203). Critically, this region modulates the troponin/tropomyosin complex's position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca2+ conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker's positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.

Epiretinal proliferation after rhegmatogenous retinal detachment.

PURPOSE: To determine the characteristics and appearance rate of epiretinal proliferation (ERP) on SD-OCT after surgery for rhegmatogenous retinal detachment (RRD) repair. METHODS: One hundred eight eyes of 108 patients who underwent one or more surgeries for RRD were enrolled. The eyes with other maculopathies that were directly related to RRD were excluded. Image acquisition was performed with SD-OCT (Heidelberg Engineering, Germany). Clinical charts were reviewed to assess clinical and surgical findings. Statistical analyses were performed using XLSTAT (Assinsoft, Paris, France). RESULTS: ERP was found in 9.3% eyes (n = 10). The mean initial visual acuity (logMAR) was 1.34 ± 0.82 in the ERP group compared to 0.49 ± 0.70 in the non-ERP group. PVR was present in 70.0% and chronic macular edema was found in 80.0% of eyes which developed ERP. The mean number of vitreoretinal surgeries in eyes with ERP was 3.3 ± 1.19 and only 1.44 ± 1.02 in eyes without. Silicone oil was used in 60.0% of eyes which developed ERP compared to 13.9% in the non-ERP group. CONCLUSION: ERP is a late-onset postoperative finding in eyes with RRD and can occur in absence of macular holes. Overall, ERP is more frequent in eyes with complicated courses of RRD including multiple operations, PVR, usage of silicone oil, and chronic macular edema.

RETINAL DETACHMENT IN EYES WITH BOSTON TYPE 1 KERATOPROSTHESIS: Surgical Techniques and Mid-Term Outcomes.

PURPOSE: To evaluate the mid-term outcomes of pars plana vitrectomy performed for retinal detachment (RD) repair after Boston Type 1 keratoprosthesis (KPro) implantation. METHODS: Retrospective review of medical records of KPro implanted at the Stein Eye Institute presenting with RD and treated by pars plana vitrectomy. Functional success was defined as a postoperative visual acuity maintained within 2 Snellen lines of the corrected distance visual acuity measured before the development of the RD (baseline) and anatomical success as an attached retina after the pars plana vitrectomy. Kaplan-Meyer survival analyses were performed. RESULTS: Among the 224 KPro performed, 28 (15.2%) RD were identified; of which, 21 (9.4%) were included. The mean follow-up was 42.5 ± 27.3 months. Vitreoretinal proliferation was present in 18 of 21 eyes (85.7%). Surgical techniques were adapted to the complex anterior segment anatomy of KPro eyes. Anatomical success was achieved in 18 of 21 eyes (85.7%). Functional success occurred in 17 of 21 eyes (81.0%), and 5 of 21 eyes (23.8%) reached 20/400 or better visual acuity at the final follow-up. The KPro was retained in 11 in 21 eyes (52.4%). The retention rate decreased from 94.7% at 1 year to 53.5% at 5 years. The most frequent complications were retroprosthetic membrane (47.6%) and corneal melt (23.8%). CONCLUSION: Modified pars plana vitrectomy techniques resulted in relatively good mid-term anatomical, functional, and retention rate outcomes, given the severity of RD at presentation and the numerous preoperative comorbidities of KPro eyes.

Clinical characteristics and visual outcomes of non-resolving subretinal fluid in neovascular AMD despite continuous monthly anti-VEGF injections: a long-term follow-up.

PURPOSE: To describe the clinical characteristics and visual outcomes of neovascular age-related macular degeneration (NV-AMD) patients with irregular pigment epithelium detachment (PED) and non-resolving subretinal fluid (SRF) despite continuous monthly injections of anti-vascular endothelial growth factor (VEGF). METHODS: This is a retrospective case series, including NV-AMD patients treated in a tertiary academic practice. Inclusion criteria were NV-AMD diagnosis, with irregular PED, and non-resolving SRF treated with continuous monthly anti-VEGF intravitreal injections. Data collection included best corrected visual acuity (BCVA), central macular thickness (CMT), sub-foveal choroidal thickness (SFCT), and type and location of PED as seen on optical coherence tomography (OCT). RESULTS: A total of 738 patients with NV-AMD underwent anti-VEGF injections during the follow-up period and 20 eyes of 19 patients (14 females and 5 males) met the inclusion criteria. Average age was 81.7 ± 6.6 years, mean follow-up time was 32.1 ± 23.5 months, and mean number of injections was 31.3 ± 24.2. Mean VA was 0.26 ± 0.21 logMAR (Snellen 20/36) at baseline versus 0.20 ± 0.23 logMAR (Snellen 20/32) at the end of the follow-up (P = 0.28). All eyes presented with sub-foveal, type 1 macular neovascularization (MNV). Average sub-foveal choroidal thickness changed from 189.70 ± 68.46 µm at baseline to 169.00 ± 63.06 µm (P < 0.001) at last follow-up. CONCLUSION: Patients with type 1 NV-AMD, irregular PED, and non-resolving SRF and under continuous treatment of monthly anti-VEGF injections may maintain good visual acuity after long period of time.

PIGMENT EPITHELIAL DETACHMENT IN AGE-RELATED MACULAR DEGENERATION: Long-Term Visual Acuity May Improve With Higher Injection Index.

PURPOSE: To define injection index (II) and assess its impact on visual acuity (VA) in pigment epithelial detachment from age-related macular degeneration over 5 years. METHODS: Injection index is defined as the mean anti-vascular endothelial growth factor injections per year from presentation. A retrospective study of 256 eyes in 213 patients was performed. Patients were stratified by II (high: ≥9, low: <9). RESULTS: Baseline characteristics showed no differences across II groups. Mean (range) follow-up, in years, was 5.02 (1.04-12.74) for all patients. Mean logMAR VA (Snellen VA) were 0.60 (20/80) and 0.56 (20/73) at baseline, 0.52 (20/66) and 0.59 (20/78) at Year 1, 0.45 (20/56) and 0.67 (20/94) at Year 2, 0.38 (20/48) and 0.66 (20/91) at Year 3, 0.41 (20/51) and 0.89 (20/155) at Year 4, and 0.35 (20/45) and 0.79 (20/123) at Year 5 for the high and low II groups, respectively. Linear regression analysis showed a gain of 0.5 approxETDRS letters with each additional injection per year. CONCLUSION: Increased II was associated with better mean VA, suggesting that long-term continuous vascular endothelial growth factor suppression may improve VA in eyes thought to carry poor prognoses.

Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels.

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13C, 15N, and nonexchangeable 2H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly (kchemlight/kchemheavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster (kchemlight/kchemheavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster (kchemlight/kchemheavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts.

FRET-based analysis of the cardiac troponin T linker region reveals the structural basis of the hypertrophic cardiomyopathy-causing Δ160E mutation.

Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. Understanding the molecular-level dysfunction elicited by TF mutations is crucial to elucidate cardiac disease mechanisms. The hypertrophic cardiomyopathy-causing cardiac troponin T (cTnT) mutation Δ160Glu (Δ160E) is located in a putative "hinge" adjacent to an unstructured linker connecting domains TNT1 and TNT2. Currently, no high-resolution structure exists for this region, limiting significantly our ability to understand its role in myofilament activation and the molecular mechanism of mutation-induced dysfunction. Previous regulated in vitro motility data have indicated mutation-induced impairment of weak actomyosin interactions. We hypothesized that cTnT-Δ160E repositions the flexible linker, altering weak actomyosin electrostatic binding and acting as a biophysical trigger for impaired contractility and the observed remodeling. Using time-resolved FRET and an all-atom TF model, here we first defined the WT structure of the cTnT-linker region and then identified Δ160E mutation-induced positional changes. Our results suggest that the WT linker runs alongside the C terminus of tropomyosin. The Δ160E-induced structural changes moved the linker closer to the tropomyosin C terminus, an effect that was more pronounced in the presence of myosin subfragment (S1) heads, supporting previous findings. Our in silico model fully supported this result, indicating a mutation-induced decrease in linker flexibility. Our findings provide a framework for understanding basic pathogenic mechanisms that drive severe clinical hypertrophic cardiomyopathy phenotypes and for identifying structural targets for intervention that can be tested in silico and in vitro.

Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase.

Heavy-enzyme isotope effects (15N-, 13C-, and 2H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (kchemlight/kchemheavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (kchemlight/kchemheavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.

Optimization of the Turnover in Artificial Enzymes via Directed Evolution Results in the Coupling of Protein Dynamics to Chemistry.

The design of artificial enzymes is an emerging field of research. Although progress has been made, the catalytic proficiency of many designed enzymes is low compared to natural enzymes. Nevertheless, recently Hilvert et al. ( Nat. Chem. 2017, 9, 50-56) created a series of five artificial retro-aldolase enzymes via directed evolution, with the final variant exhibiting a rate comparable to the naturally occurring enzyme fructose 1,6 bisphosphate aldolase. We present a study of this system in atomistic detail that elucidates the effects of mutational changes on the chemical step. Transition path sampling is used to create ensembles of reactive trajectories, and committor analysis is used to identify the stochastic separatrix of each ensemble. The application of committor distribution analysis to constrained trajectories allows the identification of changes in important protein motions coupled to reaction across the generated series of the artificial retro-aldolases. We observed two different reaction mechanisms and analyzed the role of the residues participating in the reaction coordinate of each enzyme. However, only in the most evolved variant we identified a fast motion that promotes catalysis, suggesting that this rate promoting vibration was introduced during directed evolution. This study provides further evidence that protein dynamics must be taken into account in designing efficient artificial enzymes.

Atomic resolution probe for allostery in the regulatory thin filament.

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca(2+) binding strength and the energy required to remove Ca(2+) from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca(2+) dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca(2+) ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.

Directed Evolution as a Probe of Rate Promoting Vibrations Introduced via Mutational Change.

In this article, we study with transition path sampling and reaction coordinate analysis how directed evolution in the Kemp eliminase family of artificial enzymes makes differential use of rapid rate promoting vibrations as a component of their chemical mechanism. Even though this family was initially created by placing the expected active site in a fixed protein matrix, we find a shift from largely static to more dynamic active sites that make use of donor-acceptor compression as the evolutionary process proceeds. We see that this introduction of dynamics significantly shifts the order of processes in the reaction. We also suggest that the lack of "design for dynamics" may help explain the relatively low proficiency of such designed enzymes.

Promoting Vibrations and the Function of Enzymes. Emerging Theoretical and Experimental Convergence.

A complete understanding of enzyme catalysis requires knowledge of both transition state features and the detailed motions of atoms that cause reactant molecules to form and traverse the transition state. The seeming intractability of the problem arises from the femtosecond lifetime of chemical transition states, preventing most experimental access. Computational chemistry is admirably suited to short time scale analysis but can be misled by inappropriate starting points or by biased assumptions. Kinetic isotope effects provide an experimental approach to transition state structure and a method for obtaining transition state analogues but, alone, do not inform how that transition state is reached. Enzyme structures with transition state analogues provide computational starting points near the transition state geometry. These well-conditioned starting points, combined with the unbiased computational method of transition path sampling, provide realistic atomistic motions involved in transition state formation and passage. In many, but not all, enzymatic systems, femtosecond local protein motions near the catalytic site are linked to transition state formation. These motions are not inherently revealed by most approaches of transition state theory, because transition state theory replaces dynamics with the statistics of the transition state. Experimental and theoretical convergence of the link between local catalytic site vibrational modes and catalysis comes from heavy atom ("Born-Oppenheimer") enzymes. Fully labeled and catalytic site local heavy atom labels perturb the probability of finding enzymatic transition states in ways that can be analyzed and predicted by transition path sampling. Recent applications of these experimental and computational approaches reveal how subpicosecond local catalytic site protein modes play important roles in creating the transition state.

Mechanistic Insights on Human Phosphoglucomutase Revealed by Transition Path Sampling and Molecular Dynamics Calculations.

Human α-phosphoglucomutase 1 (α-PGM) catalyzes the isomerization of glucose-1-phosphate into glucose-6-phosphate (G6P) through two sequential phosphoryl transfer steps with a glucose-1,6-bisphosphate (G16P) intermediate. Given that the release of G6P in the gluconeogenesis raises the glucose output levels, α-PGM represents a tempting pharmacological target for type 2 diabetes. Here, we provide the first theoretical study of the catalytic mechanism of human α-PGM. We performed transition-path sampling simulations to unveil the atomic details of the two catalytic chemical steps, which could be key for developing transition state (TS) analogue molecules with inhibitory properties. Our calculations revealed that both steps proceed through a concerted SN 2-like mechanism, with a loose metaphosphate-like TS. Even though experimental data suggests that the two steps are identical, we observed noticeable differences: 1) the transition state ensemble has a well-defined TS region and a late TS for the second step, and 2) larger coordinated protein motions are required to reach the TS of the second step. We have identified key residues (Arg23, Ser117, His118, Lys389), and the Mg2+ ion that contribute in different ways to the reaction coordinate. Accelerated molecular dynamics simulations suggest that the G16P intermediate may reorient without leaving the enzymatic binding pocket, through significant conformational rearrangements of the G16P and of specific loop regions of the human α-PGM.

ULTRAWIDEFIELD AUTOFLUORESENCE IN ABCA4 STARGARDT DISEASE.

PURPOSE: To report the ultrawidefield fundus autofluorescence (UWF-FAF) patterns in ABC4A Stargardt disease. METHODS: A retrospective cohort study of patients with a clinical diagnosis of Stargardt disease, confirmed ABCA4 genotype, and ultrawidefield fundus autofluorescence imaging using an Optos P200Tx. Four independent graders evaluated the images. Ultrawidefield fundus autofluorescence images were evaluated for the presence of posterior pole and peripheral findings, and were classified into one of three types (Type I: lesions confined to the macula with no peripheral findings; Type II: macular atrophy with flecks only in the periphery; Type III: macular atrophy and varying degrees of peripheral atrophy). RESULTS: Ultrawidefield fundus autofluorescence was performed on 58 eyes of 29 patients. Reviews of images revealed the presence of peripheral (outside the 55° view of standard nonwidefield FAF imaging) alterations on UWF-FAF in 76% of eyes. Overall, the UWF-FAF pattern was classified as Type I in 24% eyes (14/58), Type II in 24% (14/58), and Type III in 52% (30/58). The most common genetic mutations were c.2588G>C (6/29 patients, 20.7%), and c.5882G>A (5/29 patients, 17.2%). CONCLUSION: Ultrawidefield fundus autofluorescence reveals peripheral changes in the majority of patients with Stargardt disease. Peripheral FAF changes may have implications for diagnosis, prognosis, and management of individual patients with Stargardt disease.

EARLY DETECTION OF RETINAL HEMANGIOBLASTOMAS IN VON HIPPEL-LINDAU DISEASE USING ULTRA-WIDEFIELD FLUORESCEIN ANGIOGRAPHY.

PURPOSE: To study the use of ultra-widefield fluorescein angiography (UWF FA) in the detection and management of retinal capillary hemangioblastomas in patients with von Hippel-Lindau disease. METHODS: This is a retrospective study of patients with von Hippel-Lindau disease who underwent UWF FA using the Optos camera at a single center from June 2009 to May 2015. The clinical use of UWF FA was reviewed, and the number of hemangioblastomas identified on UWF FA was compared with ophthalmoscopy and a simulated seven standard field (7SF) FA montage. RESULTS: Twenty eyes of 10 patients were identified. Only 33% of lesions seen on UWF FA were also found on ophthalmoscopy, and 88% of lesions visualized on UWF FA were located outside the 7SF overlay. In 5 eyes that had gaze steering, 18% of lesions could be visualized only on gaze-steered images. For the 14 eyes with data available, 6 had procedures recommended and 8 eyes observed based on data from UWF FA. One of 20 eyes had a lesion on ophthalmoscopy that was missed by imaging. CONCLUSION: Ultra-widefield FA using the Optos camera is helpful for the evaluation and management of patients with von Hippel-Lindau disease. The UWF FA with gaze steering appears to detect more hemangioblastomas than ophthalmoscopy and conventional angiography.

Structurally Linked Dynamics in Lactate Dehydrogenases of Evolutionarily Distinct Species.

We present new findings about how primary and secondary structure affects the role of fast protein motions in the reaction coordinates of enzymatic reactions. Using transition path sampling and committor distribution analysis, we examined the difference in the role of these fast protein motions in the reaction coordinate of lactate dehydrogenases (LDHs) of Apicomplexa organisms Plasmodium falciparum and Cryptosporidium parvum. Having evolved separately from a common malate dehydrogenase ancestor, the two enzymes exhibit several important structural differences, notably a five-amino acid insertion in the active site loop of P. falciparum LDH. We find that these active site differences between the two organisms' LDHs likely cause a decrease in the contribution of the previously determined LDH rate-promoting vibration to the reaction coordinate of P. falciparum LDH compared to that of C. parvum LDH, specifically in the coupling of the rate-promoting vibration and the hydride transfer. This effect, while subtle, directly shows how changes in structure near the active site of LDH alter catalytically important motions. Insights provided by studying these alterations would prove to be useful in identifying LDH inhibitors that specifically target the isozymes of these parasitic organisms.

Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap.

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Evolution of Aggregate Structure in Solutions of Anionic Monorhamnolipids: Experimental and Computational Results.

The evolution of solution aggregates of the anionic form of the native monorhamnolipid (mRL) mixture produced by Pseudomonas aeruginosa ATCC 9027 is explored at pH 8.0 using both experimental and computational approaches. Experiments utilizing surface tension measurements, dynamic light scattering, and both steady-state and time-resolved fluorescence spectroscopy reveal solution aggregation properties. All-atom molecular dynamics simulations on self-assemblies of the most abundant monorhamnolipid molecule, l-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate (Rha-C10-C10), in its anionic state explore the formation of aggregates and the role of hydrogen bonding, substantiating the experimental results. At pH 8.0, at concentrations above the critical aggregation concentration of 201 µM but below ∼7.5 mM, small premicelles exist in solution; above ∼7.5 mM, micelles with hydrodynamic radii of ∼2.5 nm dominate, although two discrete populations of larger lamellar aggregates (hydrodynamic radii of ∼10 and 90 nm) are also present in solution in much smaller number densities. The critical aggregation number for the micelles is determined to be ∼26 monomers/micelle using fluorescence quenching measurements, with micelles gradually increasing in size with monorhamnolipid concentration. Molecular dynamics simulations on systems with between 10 and 100 molecules of Rha-C10-C10 indicate the presence of stable premicelles of seven monomers with the most prevalent micelle being ∼25 monomers and relatively spherical. A range of slightly larger micelles of comparable stability can also exist that become increasing elliptical with increasing monomer number. Intermolecular hydrogen bonding is shown to play a significant role in stabilization of these aggregates. In total, the computational results are in excellent agreement with the experimental results.