Nascent Hairpins in Proteins: Identifying Turn Loci and Quantitating Turn Contributions to Hairpin Stability.

Many factors influence the stability of hairpins that could appear as foldons in partially folded states of proteins; of these, the propensity of certain amino acid sequences to favor conformations that serve to align potential ß-strands for antiparallel association is likely the dominant feature. Quantitating turn propensities is viewed as the first step in developing an algorithm for locating nascent hairpins in protein sequences. Such nascent hairpins can serve to accelerate protein folding or, if they represent structural elements that differ from the final folded state, as kinetic traps. We have measured these "turn propensities" for the two most common turn types using a series of model peptide hairpins with four- and six-residue loops connecting the associated ß-strands. Loops of four to six residues with specific turn sequences containing only natural l-amino acids and glycine can provide as much as 15 kJ/mol of hairpin stabilization versus loops lacking the defined turn loci. Single-site mutations within some of the optimal connecting loops can have ΔΔG effects as large as 9-10 kJ/mol on hairpin stability. In contrast to the near universal II'/I' turns of model hairpins, a number of hairpin-supporting XZZG sequence ß-turns with αR and/or γR configurations at the ZZ unit were found. A series of turn replacements (four-residue ß-turns replaced by sequences that favor five- and six-residue reversing loops) using identical strands in our model systems have confirmed that several sequences have intrinsic turn propensities that could favor ß-strand association in a non-native strand register and thus serve as kinetic traps. These studies also indicate that aryl residues immediately flanking a turn sequence can alter relative turn propensities by as much as 9-11 kJ/mol and will need to be a part of any nascent hairpin recognition algorithm.

Peptide Inhibitors of the amyloidogenesis of IAPP: verification of the hairpin-binding geometry hypothesis.

Versions of a previously discovered ß-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at substoichiometric concentrations relative to this amyloidogenic peptide. The hairpin-binding hypothesis stands confirmed.

Engineered clathrin nanoreactors provide tunable control over gold nanoparticle synthesis and clustering.

The use of biomolecules to direct nanomaterial synthesis has been an area of growing interest due to the complexity of structures that can be achieved in naturally occurring systems. We previously reported the functionalization of self-assembled clathrin protein cages to enable synthesis of nanoparticles from a range of inorganic materials. Here, we investigate the ability of this engineered biomolecule complex to act as a tunable nanoreactor for the formation of different arrangements of gold nanoparticles in three dimensions. We find that self-assembled clathrin cages functionalized with engineered bi-functional peptides induce formation of gold nanoparticles to generate solutions of either dispersed or clustered gold nanoparticles on demand. The 3D arrangement of nanoparticles is dependent on the concentration of the engineered peptide, which fulfills multiple roles in the synthesis process including stabilization of the nanoparticle surface and localization of the nanoparticles within the self-assembled clathrin cage. We propose and evaluate a mechanism that allows us to predict the peptide concentration at which the nanoreactor behavior switches. This work provides insight into peptide-based surfactants and the potential for incorporating them into strategies for tuning biological mineralization processes in mild solution conditions to generate complex structures.

Template engineering through epitope recognition: a modular, biomimetic strategy for inorganic nanomaterial synthesis.

Natural systems often utilize a single protein to perform multiple functions. Control over functional specificity is achieved through interactions with other proteins at well-defined epitope binding sites to form a variety of functional coassemblies. Inspired by the biological use of epitope recognition to perform diverse yet specific functions, we present a Template Engineering Through Epitope Recognition (TEThER) strategy that takes advantage of noncovalent, molecular recognition to achieve functional versatility from a single protein template. Engineered TEThER peptides span the biologic-inorganic interface and serve as molecular bridges between epitope binding sites on protein templates and selected inorganic materials in a localized, specific, and versatile manner. TEThER peptides are bifunctional sequences designed to noncovalently bind to the protein scaffold and to serve as nucleation sites for inorganic materials. Specifically, we functionalized identical clathrin protein cages through coassembly with designer TEThER peptides to achieve three diverse functions: the bioenabled synthesis of anatase titanium dioxide, cobalt oxide, and gold nanoparticles in aqueous solvents at room temperature and ambient pressure. Compared with previous demonstrations of site-specific inorganic biotemplating, the TEThER strategy relies solely on defined, noncovalent interactions without requiring any genetic or chemical modifications to the biomacromolecular template. Therefore, this general strategy represents a mix-and-match, biomimetic approach that can be broadly applied to other protein templates to achieve versatile and site-specific heteroassemblies of nanoscale biologic-inorganic complexes.

Designed hairpin peptides interfere with amyloidogenesis pathways: fibril formation and cytotoxicity inhibition, interception of the preamyloid state.

Hairpin peptides bearing cross-strand Trp-Trp and Tyr-Tyr pairs at non-H-bonded strand sites modulate the aggregation of two unrelated amyloidogenic systems, human pancreatic amylin (hAM) and α-synuclein (α-syn), associated with type II diabetes and Parkinson's disease, respectively. In the case of hAM, we have previously reported that inhibition of amyloidogenesis is observed as an increase in the lag time to amyloid formation and a diminished thioflavin (ThT) fluorescence response. In this study, a reduced level of hAM fibril formation is confirmed by transmission electron microscopy imaging. Several of the hairpins tested were significantly more effective inhibitors than rat amylin. Moreover, a marked inhibitory effect on hAM-associated cytotoxicity by the more potent hairpin peptide is demonstrated. In the case of α-syn, the dominant effect of active hairpins was, besides a weakened ThT fluorescence response, the earlier appearance of insoluble aggregates that do not display amyloid characteristics with the few fibrils observed having abnormal morphology. We attribute the alteration of the α-synuclein aggregation pathway observed to the capture of a preamyloid state and diversion to nonamyloidogenic aggregates. These ß-hairpins represent a new class of amyloid inhibitors that bear no sequence similarity to the amyloid-producing polypeptides that are inhibited. A mechanistic rationale for these effects is proposed.

Very short peptides with stable folds: building on the interrelationship of Trp/Trp, Trp/cation, and Trp/backbone-amide interaction geometries.

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i-2 Trp ring, a particularly stable (DeltaG(U) > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH(2), results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed "edge-to-face" (EtF) Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater beta-propensity of Trp. The pi-cation, and Trp/Gly-H(N) interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly-H(N) in -WX(n)G- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred "face" residue in aryl/aryl pairings, presumably because of its greater pi basicity.

Identification of XAF1 as an antagonist of XIAP anti-Caspase activity.

The inhibitors of apoptosis (IAPs) suppress apoptosis through the inhibition of the caspase cascade and thus are key proteins in the control of cell death. Here we have isolated the protein XIAP-associated factor 1 (XAF1) on the basis of its ability to bind XIAP, a member of the IAP family. XIAP suppresses caspase activation and cell death in vitro, and XAF1 antagonizes these XIAP activities. Expression of XAF1 triggers a redistribution of XIAP from the cytosol to the nucleus. XAF1 is ubiquitously expressed in normal tissues, but is present at low or undetectable levels in many different cancer cell lines. Loss of control over apoptotic signalling is now recognized as a critical event in the development of cancer. Our results indicate that XAF1 may be important in mediating the apoptosis resistance of cancer cells.

Extrapolation of measures of work for surveyed services to other services.

A national survey of physicians produced detailed data on the work involved in performing 372 different services. This article describes methods developed to extrapolate the study data to a larger universe of services, defined by the Physicians' Current Procedural Terminology, edition 4. Because data measuring work inputs for nonsurveyed services presently are unavailable, we devised an extrapolation method that makes use of available charge data without building their inherent distortions into the extrapolated scale. To neutralize the effect of these distortions, we used small, homogeneous families of services as the basic units for the extrapolations and assumed that charges are reasonable indicators of relative work within such families. To produce extrapolated work values within each family, we multiplied an estimate of work based on survey data for a benchmark procedure by charge-based ratios that represent the relationships between surveyed and nonsurveyed services. These extrapolations can be used in constructing a Resource-Based Relative Value Scale.

Results, potential effects, and implementation issues of the Resource-Based Relative Value Scale.

This article presents the overall results of the Resource-Based Relative Value Scale (RBRVS) study. We present resource-based relative values for selected services in each of the 18 specialties we studied. We found that preservice and postservice work represents close to 50% of total work for invasive services and 33% of total work for evaluation/management services. We also found that the work per unit time (a measure of intensity) for invasive services is about three times that of evaluation/management. We developed a simple model and simulated an RBRVS-based fee schedule for the Medicare program under a "budget-neutral" assumption. Results for 30 commonly performed services show that office visit fees for evaluation/management services could rise by 70%, while some surgical fees could drop by 60%. We also simulated what the Medicare outlays would have been in 1986 for categories of medical services under an RBRVS-based fee schedule. We found that total Medicare payments for evaluation/management services would have increased by about 56%. Invasive, imaging, and laboratory services would have decreased by 42%, 30%, and 5%, respectively. We also discuss implementation issues related to an RBRVS-based fee schedule, such as the determination of a monetary conversion factor, practice costs, billing codes, and the need to evaluate the potential impacts of an RBRVS-based payment system on the cost and quality of health care.

Comparative trends in hospital expenses, finances, utilization, and inputs, 1970-81.

The annual surveys of the American Hospital Association historically have been only national source of statistics on hospital structure and performance. Although valuable, this source has not provided the policy or research community with hospital-specific information on revenues, assets, and financial status. Data on these and other variables from heretofore unpublished Medicare cost report data are presented in this article. Hospital expenses, revenues, profits, indebtedness, utilization, investments, and employees are trended over the 1970-81 period by urban-rural location, teaching status, and ownership. It is indicated in these data that a major transformation in the hospital industry has occurred in response to cost-based Medicare-Medicaid and other factors that made acute care essentially unaffordable to the average citizen. The health maintenance organization movement and Medicare's prospective payment system are seen as logical reactions to this transformation.

The economic costs of smoking and benefits of quitting for individual smokers.

The results of a study that estimated the expected lifetime economic consequences of cigarette smoking for individual smokers are reported herein. The estimates were obtained by combining age- and sex-specific estimates of the incidence-based costs of three smoking-related diseases (lung cancer, coronary heart disease, and emphysema) with estimates of smokers' increased likelihood of developing these illnesses in each remaining year of life relative to nonsmokers. Estimates of the economic consequences of quitting based on these disease cost estimates and on estimates of exsmokers' probability of future disease relative to continuing smokers are also reported. Both the estimates of the economic costs of smoking and the benefits of quitting were calculated separately for men and women between the ages of 35 and 79 who were light, moderate, or heavy cigarette smokers. While the economic costs of smoking varied considerably by sex, age, and amount smoked, they were significant for all groups of smokers. Costs for a 40-year-old man, for example, ranged from $20,000 for a smoker of less than one pack of cigarettes per day to over $56,000 for a smoker of more than two packs of cigarettes per day. The economic benefits of quitting also were found to be sizable for all groups of smokers.

A four-cell typology to measure hospital teaching status.

Hospital participation in graduate medical education is of increasing interest in the health policy arena because of its financial and behavioral implications for hospitals. In this attempt to create a more refined method of measuring hospital teaching status, a four-cell classification, or typology, of all nonfederal short-term general teaching hospitals was developed from 11 descriptors of educational output. This typology proved to be both effective in distinguishing different types of teaching institutions and useful in analyzing interhospital variation in scope of services, case mix, and cost.

Medicare benefits: a reassessment.

Changes in beneficiary cost-sharing provisions must be part of any multi-faceted strategy for Medicare's fiscal solvency . The current flawed benefit structure is seen as inefficient, inequitable , and contrary to prudent insurance principles. An income-related and selective increase in cost-sharing, combined with maximum liability, is proposed.