Shawn Y. Stevens

Structural insights into substrate binding by the molecular chaperone DnaK

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 α-helical domain, DnaK(1–507), retains the ability to support λ 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 β3 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 β-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the β-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity.

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.

The solution structure of the bacterial HSP70 chaperone protein domain DnaK(393–507) in complex with the peptide NRLLLTG

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 α‐helical domain that characterizes earlier published peptide‐bound structures of the Hsp70s. It is established that removing the α‐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. 15N‐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.

Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase

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.

Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies

We have demonstrated that the gel shift assay, a powerful method to study protein.DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA.DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein.DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels which are known to form non-restrictive matrices, do not perturb anti-DNA.DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measured in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.

A middle school instructional unit for size and scale contextualized in nanotechnology

Abstract Size and scale is a “big idea” in nanoscale science and engineering and is poorly understood by secondary students. This paper describes the design process, implementation, and evaluation of a 12-h instructional unit for size and scale, in a summer science camp for middle school students from a low SES public school district. Instructional activities were designed following a construct-centered design approach and included the use of microscopes, custom-made computer simulations, and 2-D and 3-D scale models. The unit followed a project-based instructional approach and was contextualized with the driving question, “How can nanotechnology keep me from getting sick?” Pre- and post-intervention interviews revealed that students significantly increased their qualitative and quantitative knowledge of the size of objects including atom, viruses, and cells, with an effect size of 0.8 for an overall metric. The campers closed the gap with private middle school students and on some measures surpassed high school students from the same district. The principle of “broad spectrum” curriculum and instruction – activities that target specific advanced understandings but simultaneously scaffold or support the learning of more fundamental, prerequisite ideas – was inductively generated from an analysis of the learning activities.

Learning progressions as a guide for developing meaningful science learning: A new framework for old ideas

The development of learning progressions is one approach for creating the types of coher ent curriculum frameworks that have been identified as predictors for high-performing scores on international stem assessments. We have developed a learning progression that describes how secondary students may build more sophisticated understanding of the struc ture, properties, and behavior of matter, and that also outlines the connections and relation ships among ideas needed to develop more expert understanding. We used data collected from 82 individual interviews with secondary students and from assessments administered to 4000 Us middle school students to characterize how learners select and apply ideas to explain a range of transformation of matter phenomena. We found that most students relied on a limited set of ideas in their explanations, but that with the proper support, even middle school students were able to appropriately integrate ideas involving the structure of matter, conservation, interactions, and energy to provide mechanistic explanations of transforma tion phenomena.

Tracking student learning over time using constructcentred design

This introductory collection brings together contributions from a range of international researchers, each working within their own traditions, to explore current perspectives on the use of analytical approaches in education. Providing a close examination of cutting-edge data analysis techniques, each contribution describes a set of tools to assist prospective researchers as they decide which analytical approach or approaches best suit their own research endeavours.