Amyloid fibers are aggregated, yet highly ordered, beta-sheet-rich assemblies of misfolded proteins. Order is established in such systems following profiles indicative of nucleation-dependent assembly. Nucleation dependence suggests that specific interactions, such as long-range contacts and/or strand registration, are critical to establishing initial fiber structure. Here, we show that amino acids at selected positions participate in key interactions that modulate the pathway of amyloid fiber formation by the hormone, islet amyloid polypeptide (IAPP). Specifically, we investigated the role of amide side-chain interactions in the process of IAPP assembly. We mutated five of the asparagine side chains in IAPP and assessed their effects on the kinetics of assembly. We find that the asparagine amide side chains strongly dictate the ability of IAPP to form fibers. In particular, the elimination of two specific asparagines results in near and total loss of amyloid, respectively. Interestingly, the two asparagines are located in a recently identified domain with alpha-helical bias. These sensitivities are unusual for IAPP, as IAPP is generally tolerant to mutation. Here, we demonstrate this mutational tolerance by assessing 10 alterations at five distinct sites. In all cases, the constructs form fibers on timescales perturbed by less than a factor of two compared with wild-type protein. These findings indicate the presence of key specific interactions that are the determinants of IAPP amyloid formation.
Amyloidogenesis from soluble protein requires conformational and oligomeric assembly steps. In systems where the precursor protein is natively unfolded, such as islet amyloid polypeptide (IAPP), forces and structural changes relevant to protein unfolding are not thought to participate in the assembly mechanism. Thus, fiber core structure elements should provide the dominant contributions to assembly kinetics. Here we show, however, that residues outside the amyloid core can influence the mechanism of IAPP fiber assembly. IAPP possesses an intramolecular disulfide bond between residues 2 and 7. This short-range disulfide prohibits the N-terminal region from adopting the -strand structure of an amyloid. We examined the role of this disulfide in fiber formation by generating a truncated construct ) and a stable reduced form of the full-length protein (IAPP CAM ). The fiber structures and assembly kinetics of these variants were assessed via optical and mass spectroscopy. Our data confirm that the disulfide does not contribute to the amyloid fiber core structure. Remarkably, however, it plays a central role in the assembly mechanism. First, loss of the disulfide substantially reduces fiber formation by secondary nucleation, i.e., the ability of pre-existing fibers to participate in the formation of new fibers. Second, the bypass of nucleation by seed addition is a two-step process, termed activation. Loss of the disulfide eliminates this two-step nature of seeded kinetics.Keywords: amylin; amyloid; islet amyloid polypeptide; protein folding; secondary nucleation, phasemediated fibrillogenesis; type II diabetes The oligomerization of soluble protein into large, highly ordered fibrillar structures, termed amyloids, is a common feature of a number of diseases including, for example, Alzheimer's disease and dialysis-related amyloidosis (Rochet and Lansbury 2000). Interestingly, proteins that undergo amyloid fiber formation vary greatly in their native structures and functions, but in the fiber state, exhibit common features. Amyloid fibers form a core that is predominantly composed of -sheets. The strands within these sheets are organized perpendicular to the long axis of the fiber with backbone hydrogen bonding oriented in parallel to the fiber axis (Eanes and Glenner 1968; Sunde and Blake 1997). The common core structure of amyloids suggests that assembly mechanisms across different systems are comparable. Indeed, the overall kinetic behaviors of amyloids also share common features. A protein placed under amyloidogenic conditions will initially remain soluble. This quiescent phase, termed the lag phase, is followed by collective assembly into the aggregated state. This implies the existence of nucleation processes akin to crystallization (Harper and Lansbury 1997). As with crystallization, nucleation reactions within the lag phase may be bypassed by providing exogenous fiber from a previously conducted reaction. A number of intermediate states likely participate in the assembly process. As amyloidogenesis o...
Prestin is the motor protein within the lateral membrane of outer hair cells (OHCs), and it is required for mammalian cochlear amplification. Expression of prestin precedes the onset of hearing in mice, and it has been suggested that prestin undergoes a functional maturation within the membrane coincident with the onset of hearing. We have developed a tetracycline-inducible prestin-expressing cell line that we have used to model prestin's functional maturation. We used prestin's voltage-dependent nonlinear charge movement (or nonlinear capacitance) as a test of function and correlated it to biochemical measures of prestin expressed on the cell surface. An initial stage of slow growth in charge density is accompanied by a rapid increase in our estimate of charge carried by an individual motor. A rapid growth in charge density follows and strongly correlates with an increasing ratio between an apparently larger and smaller monomer, suggesting that the latter exerts a dominant-negative effect on function. Finally, there is a gradual depolarizing shift in the voltage of peak capacitance, similar to that observed in developing OHCs. This inducible system offers many opportunities for detailed studies of prestin.
While the impact of authentic research experiences in STEM on student engagement and interest in science has been documented, less is known about the role of peer communities in fostering this interest and engagement. This research explores the idea that a strong peer community can catalyze deep learning and engagement in scientific research among high school students. The program engaged 20 high school students in a year-long community-based participatory research project in public health each year. The study used a mixed methods approach, combining data from focus group discussions, observations, and surveys to describe the program's impact on participants. Analysis across three years reveals that (a) the program was associated with a statistically significant shift in students' identity as researchers, with a medium growth effect size (Cohen's d) for the second and third years, which moderated by the end of the program, and (b) the peer community played a central role in the participants' engagement in the program, on their identity as researchers, and strengthened their interest in STEM. These findings convey the importance of designing STEM experiences that build strong peer communities around science practices and how such communities can have profound impacts on students' identities in STEM.
The Likert item response format for items is almost ubiquitous in the social sciences and has particular virtues regarding the relative simplicity of item-generation and the efficiency for coding responses. However, in this article, we critique this very common item format, focusing on its affordance for interpretation in terms of internal structure validity evidence. We suggest an alternative, the Guttman response format, which we see as providing a better approach for gathering and interpreting internal structure validity evidence. Using a specific survey-based example, we illustrate how items in this alternative format can be developed, exemplify how such items operate, and explore some comparisons between the results from using the two formats. In conclusion, we recommend usage of the Guttman response format for improving the interpretability of the resulting outcomes. Finally, we also note how this approach may be used in tandem with items that use the Likert response format to help balance efficiency with interpretability.
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