High pressure NMR reveals conformational perturbations by disease-causing mutations in amyloid ß-peptide.

Here we present the high pressure NMR characterization of Aß42 and two Aß40 variants with Alzheimer-causing mutations E22G and D23N. While chemical shifts only identified localized changes at ambient pressure compared with Aß40, high pressure NMR revealed a common site with heightened pressure sensitivity at Q15, K16 and L17 in all three variants, which correlates to higher ß-propensity at central hydrophobic cluster (CHC) and faster aggregation.

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol-1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ΔVf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.

Characterization of Aß Monomers through the Convergence of Ensemble Properties among Simulations with Multiple Force Fields.

Amyloid ß (Aß) monomers represent a base state in the pathways of aggregation that result in the fibrils and oligomers implicated in the pathogenesis of Alzheimer's disease (AD). The structural properties of these intrinsically disordered peptides remain unclear despite extensive experimental and computational investigations. Further, there are mutations within Aß that change the way the peptide aggregates and are known to cause familial AD (FAD). Here, we analyze the ensembles of different isoforms (Aß42 and Aß40) and mutants (E22Δ, D23N, E22K, E22G, and A2T in Aß40) of Aß generated with all-atom replica exchange molecular dynamics (REMD) simulations on the µs/replica time scale. These were run using three different force field/water model combinations: OPLS-AA/L and TIP3P ("OPLS"), AMBER99sb-ILDN and TIP4P-Ew ("ILDN"), as well as CHARMM22* and TIP3SP ("CHARMM"). Despite fundamental changes in simulation parameters, we find that the resulting ensembles demonstrate a strong convergence in structural properties. In particular, antiparallel contacts between L17-A21 and A30-L34 are prevalent in ensembles of Aß40, directly forming ß sheets in the OPLS and ILDN combinations. A21-A30 commonly forms an interceding region that rarely interacts with the rest of the peptide. Further, Aß42 contributes new ß hairpin motifs involving V40-I41 in both OPLS and ILDN. However, the structural flexibility of the central region and the electrostatic interactions that characterize it are notably different between the different conditions. Further, for OPLS, each of the FAD mutations disrupts central bend character and increases the polymorphism of antiparallel contacts across the central region. However, the studied mutations in the ILDN set primarily encourage more global contacts involving the N-terminus and the central region, and promote the formation of new ß topologies that may seed different aggregates involved in disease phenotypes. These differences aside, the large degree of agreement between simulation sets across multiple force fields provides a generalizable characterization of Aß that is also consistent with experimental data and models.

Familial Alzheimer's mutations within APPTM increase Aß42 production by enhancing accessibility of ε-cleavage site.

The high Aß42/Aß40 production ratio is a hallmark of familial Alzheimer's disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aß is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ε-cleavage at either T48 or L49, resulting in subsequent production of Aß42 or Aß40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ε-cleavage, and consequently shift cleavage preference towards Aß42.

Green-lighting green fluorescent protein: faster and more efficient folding by eliminating a cis-trans peptide isomerization event.

Wild-type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis-trans peptide bond isomerization. The only conserved cis-peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X-P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans-peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.

Aß monomers transiently sample oligomer and fibril-like configurations: ensemble characterization using a combined MD/NMR approach.

Amyloid ß (Aß) peptides are a primary component of fibrils and oligomers implicated in the etiology of Alzheimer's disease (AD). However, the intrinsic flexibility of these peptides has frustrated efforts to investigate the secondary and tertiary structure of Aß monomers, whose conformational landscapes directly contribute to the kinetics and thermodynamics of Aß aggregation. In this work, de novo replica exchange molecular dynamics (REMD) simulations on the microseconds-per-replica timescale are used to characterize the structural ensembles of Aß42, Aß40, and M35-oxidized Aß42, three physiologically relevant isoforms with substantially different aggregation properties. J-coupling data calculated from the REMD trajectories were compared to corresponding NMR-derived values acquired through two different pulse sequences, revealing that all simulations converge on the order of hundreds of nanoseconds-per-replica toward ensembles that yield good agreement with experiment. Though all three Aß species adopt highly heterogeneous ensembles, these are considerably more structured compared to simulations on shorter timescales. Prominent in the C-terminus are antiparallel ß-hairpins between L17-A21, A30-L36, and V39-I41, similar to oligomer and fibril intrapeptide models that expose these hydrophobic side chains to solvent and may serve as hotspots for self-association. Compared to reduced Aß42, the absence of a second ß-hairpin in Aß40 and the sampling of alternate ß topologies by M35-oxidized Aß42 may explain the reduced aggregation rates of these forms. A persistent V24-K28 bend motif, observed in all three species, is stabilized by buried backbone to side-chain hydrogen bonds with D23 and a cross-region salt bridge between E22 and K28, highlighting the role of the familial AD-linked E22 and D23 residues in Aß monomer folding. These characterizations help illustrate the conformational landscapes of Aß monomers at atomic resolution and provide insight into the early stages of Aß aggregation pathways.

Tranilast binds to aß monomers and promotes aß fibrillation.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer's disease (AD), in which amyloid ß-protein (Aß) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aß40 monomers with ∼300 µM affinity. Remarkably, tranilast increases Aß40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aß monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aß-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

Eight years of the Mayo International Health Program: what an international elective adds to resident education.

OBJECTIVE: To examine the educational benefits of international elective rotations during graduate medical education. PARTICIPANTS AND METHODS: We studied Mayo International Health Program (MIHP) participants from April 1, 2001, through July 31, 2008. Data from the 162 resident postrotation reports were reviewed and used to quantitatively and qualitatively analyze MIHP elective experiences. Qualitative analysis of the narrative data was performed using NVivo7 (QRS International, Melbourne, Australia), a qualitative research program, and passages were coded and analyzed for trends and themes. RESULTS: During the study period, 162 residents representing 20 different specialties were awarded scholarships through the MIHP. Residents rotated in 43 countries, serving over 40,000 patients worldwide. Their reports indicated multiple educational and personal benefits, including gaining experience with a wide variety of pathology, learning to work with limited resources, developing clinical and surgical skills, participating in resident education, and experiencing new peoples and cultures. CONCLUSION: The MIHP provides the structure and funding to enable residents from a variety of specialties to participate in international electives and obtain an identifiable set of unique, valuable educational experiences likely to shape them into better physicians. Such international health electives should be encouraged in graduate medical education.

Clinical consequences of withholding versus administering renin-angiotensin-aldosterone system antagonists in the preoperative period.

BACKGROUND: Hospitalists involved in perioperative care either stop or continue until the day of surgery renin-angiotensin-aldosterone system antagonists (either angiotensin-converting enzyme inhibitors [ACEI] or angiotensin II receptor subtype 1 antagonists [ARA]) in patients who use these agents chronically. This practice variation reflects uncertainty regarding the risks and benefits of either approach. PURPOSE: The purpose of this study was to assess the clinical consequences of preoperatively continuing versus withholding ACEI/ARAs in patients treated chronically with these agents. DATA SOURCES AND STUDY SELECTION: We comprehensively searched 7 major electronic databases, considered references from selected reviews, hand-searched journals, and communicated with experts. We included randomized trials and observational studies. DATA EXTRACTION: We evaluated the relative risk (RR) of hypotension requiring vasopressors and of myocardial infarction in patients who did or did not receive an immediate preoperative dose of ACEI or ARA. DATA SYNTHESIS: Random-effects meta-analysis from 5 studies totaling 434 patients suggested that patients receiving an immediate preoperative ACEI/ARA dose were more likely (RR 1.50, 95% CI 1.15-1.96) to develop hypotension requiring vasopressors at or shortly after induction of anesthesia. Sufficient data were not available to assess other outcomes. CONCLUSION: Preoperative administration of ACEI/ARAs increases intraoperative hypotension. The long-term clinical consequences of continuing versus withholding preoperative ACEI/ARAs are unknown. This uncertainty stems in part from the absence to date of randomized trials designed specifically to examine patient-important consequences of this decision.

In-room display of day and time patient is anticipated to leave hospital: a "discharge appointment".

BACKGROUND: We learned from a focus group that many patients find discharge to be one of the least satisfying elements of the hospital experience. Patients cited insufficient communication about the day and time of the impending discharge as a cause of dissatisfaction. OBJECTIVE: In partnership with the Institute for Healthcare Improvement, Improvement Action Network collaborative, we tested the practicality of an in-room "discharge appointment" (DA) display. SETTING AND PATIENTS: Eight inpatient care units in 2 hospitals at an academic medical center (Mayo Clinic, Rochester, MN). INTERVENTION: DA displayed on a specially designed bedside dry-erase board. MEASUREMENTS: The primary outcome was the proportion of discharged patients who had been given a DA, including same-day DAs. Secondary outcomes were (1) the proportion of DAs scheduled before the actual dismissal day and (2) the timeliness of the actual departure compared with the DA. RESULTS: During the 4-month period, 2046 patients were discharged. Of those, 1256 patients (61%) were given a posted DA, of which 576 (46%) were scheduled at least a day in advance and 752 (60%) departed from the care unit within 30 minutes of the appointed time. CONCLUSIONS: With a program for in-room display of a DA in various hospital units, more than half the patients had a DA set, and most of the DA patients departed on time. Further investigation is needed to determine the effect of DAs on patient and provider satisfaction.

Hospital medicine fellowships: works in progress.

The field of hospital medicine continues to grow rapidly, and with this growth has come the realization that residency alone may not provide the full complement of skills required of a successful hospitalist. As a result, several institutions have started hospitalist fellowships, new programs with the specific goal of training clinicians to improve hospital care. These fellowships offer diverse approaches to preparation for a hospitalist career, with opportunities for advanced training in clinical care, teaching, research, and quality improvement. This article provides an overview of the programs, explores the choices for trainees in selecting a fellowship, and the challenges for institutions in developing a new fellowship. Although hospitalist fellowships are still in evolution, they will play an important role in the development of hospital medicine.