We describe efforts toward the development of a hypothetical learning progression (HLP) for the growth of grade 7-14 students' models of the structure, behavior and properties of matter, as it relates to nanoscale science and engineering (NSE). This multi-dimensional HLP, based on empirical research and standards documents, describes how students need to incorporate and connect ideas within and across their models of atomic structure, the electrical forces that govern interactions at the nano-, molecular, and atomic scales, and information in the Periodic Table to explain a broad range of phenomena. We developed a progression from empirical data that characterizes how students currently develop their knowledge as part of the development and refinement of the HLP. We find that most students are currently at low levels in the progression, and do not perceive the connections across strands in the progression that are important for conceptual understanding. We suggest potential instructional strategies that may help students build organized and integrated knowledge structures to consolidate their understanding, ready them for new ideas in science, and help them construct understanding of emerging disciplines such as NSE, as well as traditional science disciplines. Recent scientific research has revealed that matter exhibits novel, often unexpected properties as it transitions between the bulk form and that of individual atoms and molecules. This transition generally occurs at the nanoscale, where at least one dimension measures between 10 À9 and 10 À7 m. Scientists and policy-makers predict that the new information and technologies resulting from nanoscale science and engineering (NSE) research will have extensive societal implications that may be realized in a broad range of areas, including health care, agriculture, food, water purification, and energy and environmental concerns (PCAST, 2005). These predictions have created a need to incorporate ideas related to NSE into the science curriculum.A foundation for NSE literacy must include a robust model of the nature of matter, which includes the structure of matter, how it behaves and interacts, as well as its properties and what determines those properties. These ideas are also the foundation of understanding chemistry and are considered important aspects of science literacy (American Association for the Advancement of Science [AAAS], 1993; National Research Council [NRC], 1996). Due to the extensive nature of the science content, we will focus on only a portion of it in this manuscript. We describe our efforts toward the development of a hypothetical learning progression (HLP) that characterizes a path along which grade 7-14 students may develop more sophisticated models of atomic structure, and the electrical forces that govern interactions at the nano-, molecular, and atomic scales. Each of these knowledge domains represents a significant portion of one or more big ideas of NSE (Stevens, Sutherland, & Krajcik, in press). We followed an iterative, desi...

Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.

The design, synthesis, and analysis of analogs of d(CGCGAATTCGCG)2 possessing one or two intrahelical disulfide cross-links is reported. The cross-linked oligomers were prepared by first synthesizing duplexes where the 3‘- and 5‘-terminal bases of the parent sequence were replaced with N 3 -thioethylthymidine. Following deprotection and purification, air oxidation afforded the desired cross-linked constructs in high yield. Analysis of both the oxidized (disulfide cross-linked) and reduced (thiol modified) duplexes by UV, circular dichroism, and NMR spectroscopies along with susceptibility to EcoRI cleavage indicates that the modifications are not structurally perturbing. Optical thermal denaturation and differential scanning calorimetry measurements suggest that introducing disulfide cross-link(s) into d(CGCGAATTCGCG)2 does, however, cause two fundamental changes. First, the cross-link(s) increase the thermal stability of the modified duplexes by changing the molecularity of denaturation without an increase in enthalpy. Second, the disulfide cross-link traps one of the conformations of the conformationally heterogeneous parent molecule resulting in a conformationally homogeneous system. Both of these features are themselves unique and will be important for further applications of disulfide cross-linked oligomers such as these in studies of nucleic acid structure and function.

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

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The Hsp70 family of molecular chaperones participates in a number of cellular processes, including binding to nascent polypeptide chains and assistance in protein (re)folding and degradation. We present the solution structure of the substrate binding domain (residues 393-507) of the Escherichia coli Hsp70, DnaK, that is bound to the peptide NRLLLTG and compare it to the crystal structure of DnaK(389-607) bound to the same peptide. The construct discussed here does not contain the ␣-helical domain that characterizes earlier published peptide-bound structures of the Hsp70s. It is established that removing the ␣-helical domain in its entirety does not affect the primary interactions or structure of the DnaK(393-507) in complex with the peptide NRLLLTG. In particular, the arch that protects the substrate-binding cleft is also formed in the absence of the helical lid.15 N-relaxation measurements show that the peptide-bound form of DnaK(393-507) is relatively rigid. As compared to the peptide-free state, the peptide-bound state of the domain shows distinct, widespread, and contiguous differences in structure extending toward areas previously defined as important to the allosteric regulation of the Hsp70 chaperones.

Anti-DNA autoantibodies are a serological hallmark of the autoimmune disorder systemic lupus erythematosus. In a process involving antigen recognition, these antibodies are also believed to mediate the kidney inflammation that results in much of the morbidity and mortality associated with lupus. However, the nature of specific DNA antigens recognized by anti-DNA and the way in which anti-DNA interact with these molecules remains poorly understood. As a first step in defining the molecular basis of anti-DNA interactions, binding site selection experiments were conducted using three clonally related murine monoclonal anti-ssDNA autoantibodies previously isolated from a lupus prone MRL-lpr mouse (Swanson, P. C., Ackroyd, P. C., and Glick, G. D. (1996) Biochemistry 35, 1624-1633). Studying the interaction of these autoantibodies with the selected sequences (and variants) through affinity measurements and footprinting experiments provides evidence for sequence-specific binding of ssDNA. However, despite the similarity in amino acid sequence between the three mAbs, only mAb 11F8 appears to possess sequence specificity. The salient features of the interaction between 11F8 and its selected sequence (e.g., limited dependence of ionic strength upon binding, cross-reactivity, and conformational complementarity) are best described by combining the paradigms invoked to explain protein.nucleic acid and antibody.antigen recognition.

The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.