About

David E. Wemmer, born in 1951, is a Professor of the Graduate School and Emeritus Professor of Chemistry at the University of California, Berkeley. He holds a B.S. degree from the University of California, Davis, obtained in 1973, and a Ph.D. in Physical Chemistry from UC Berkeley, earned in 1979. His professional experience includes serving as Operations Manager of the NIH NMR Facility at Stanford University from 1980 to 1982 and as a Research Assistant Professor at the University of Washington from 1983 to 1985. Professor Wemmer's research focuses on biophysical chemistry, specifically investigating proteins, nucleic acids, and their interactions using NMR spectroscopy. His laboratory aims to understand the interactions that govern the function of biopolymers such as proteins, DNA, and RNA, including conformational stability and molecular interactions essential for biological activity. His work employs nuclear magnetic resonance, particularly multidimensional methods with biosynthetic isotope enrichment, to determine solution structures, analyze conformational fluctuations, and study complex formation. His research has contributed to understanding DNA-ligand interactions, especially in recognizing sequence-specific binding of small molecules to DNA, and exploring the structural and functional aspects of regulatory proteins involved in genetic expression, including transcription factors and RNA-binding proteins.

Research topics

• Chemistry
• Stereochemistry
• Crystallography
• Biology
• Biochemistry

Selected publications

• Magnetic resonance imaging of living systems by remote detection

  OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-23

  articleOpen access1st authorCorresponding

  A novel approach to magnetic resonance imaging is disclosed. Blood flowing through a living system is prepolarized, and then encoded. The polarization can be achieved using permanent or superconducting magnets. The polarization may be carried out upstream of the region to be encoded or at the place of encoding. In the case of an MRI of a brain, polarization of flowing blood can be effected by placing a magnet over a section of the body such as the heart upstream of the head. Alternatively, polarization and encoding can be effected at the same location. Detection occurs at a remote location, using a separate detection device such as an optical atomic magnetometer, or an inductive Faraday coil. The detector may be placed on the surface of the skin next to a blood vessel such as a jugular vein carrying blood away from the encoded region.

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• A Saturation-Mutagenesis Analysis of the Interplay Between Stability and Activation in Ras

  bioRxiv (Cold Spring Harbor Laboratory) · 2022-01-06 · 1 citations

  preprintOpen access

  Abstract Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al . eLife , 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g., at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with the destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras – and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

  PublisherOA PDFDOI

• Author response: A saturation-mutagenesis analysis of the interplay between stability and activation in Ras

  2022-01-25

  peer-reviewOpen access

  Article Figures and data Abstract Editor's evaluation Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras – and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation. Editor's evaluation This is a well executed study that provides significant new insights and connections between protein structure, stability and function. Numerous sites of mutation are identified that activate the small GTPase Ras beyond the few cancer "hot spots" that predominate cancer genomics data. While cancer causing mutations selectively alter regulatory interactions with GTPase-activating proteins, careful biophysical analysis presented here leads to the conclusion that Ras is also activated by mutations that decrease stability (increase dynamics) short of unfolding. Thus, protein sensitivity to activating mutations can depend on the stability threshold in addition to regulatory interactions with binding partners. https://doi.org/10.7554/eLife.76595.sa0 Decision letter eLife's review process Introduction The small GTPase Ras (Figure 1A) cycles between active GTP-bound and inactive GDP-bound states (Figure 1B; Wittinghofer and Vetter, 2011). There are four principal isoforms of human Ras (H-Ras, N-Ras, and two splice variants of K-Ras; referred to collectively as 'Ras'). GTP-bound Ras binds to the Ras-binding domains (RBDs) of effector proteins, such as Raf kinases and PI-3 kinase (PI3K), triggering signaling cascades that result in cell proliferation (Ehrhardt et al., 2002; Pylayeva-Gupta et al., 2011; Schubbert et al., 2007). Ras proteins have weak intrinsic GTPase activity (Ehrhardt et al., 2002). In the cell, Ras activity is controlled by two kinds of regulators: GTPase-activating proteins (GAPs) and guanine nucleotide-exchange factors (GEFs). GAPs stimulate the hydrolysis of GTP, thereby converting Ras to the inactive GDP-bound state (Ahmadian et al., 1997). Spontaneous exchange of GDP for GTP is slow, and nucleotide exchange and re-activation of Ras is accelerated by GEFs (Bandaru et al., 2019; Boriack-Sjodin et al., 1998; Ehrhardt et al., 2002; Harrison et al., 2016; Vetter and Wittinghofer, 2001). Figure 1 with 1 supplement see all Download asset Open asset Ras G-domain, the switching cycle, and schematics of the two selection assays. (A) The three principal sites of cancer mutations in Ras, referred to as the three cancer hotspots – Gly 12, Gly 13, and Gln 61 – are shown in the K-Ras structure. The C-terminal helix extension is indicated. PDB ID: 2MSD (Mazhab-Jafari et al., 2015). (B) Ras cycles between signaling-active GTP-bound and signaling-inactive GDP-bound states. (C) The bacterial-two-hybrid system couples the C-Raf-RBD•Ras-GTP interaction to the transcription of an antibiotic resistance gene (Bandaru et al., 2017). Ras is fused to the N-terminal domain of the α-subunit of the E. coli RNA polymerase. C-Raf-RBD is fused to the λ-cI protein. The GAP and the GEF can be co-expressed in the system. E. coli cells are transformed with a DNA library of 'unselected' variants. The bacteria are grown in the presence of an antibiotic for 9 hr, then the DNA library of 'selected' variants is isolated. Next-generation sequencing (NGS) is used to count the frequency of each variant in the unselected and selected samples. (D) Ba/F3 assay for Ras activity. Mutant H-Ras libraries are transfected into HEK 293T cells to generate a retroviral library of mutants. Ba/F3 cells are transduced with the retroviral library. After 24 hours, a fraction of the cells are used as the unselected population, and the remainder of the cells – the selected population – are cultured for 7 days in the absence of IL-3 before harvesting them by centrifugation. The genomic DNA of the selected and unselected populations is isolated and sequenced using NGS. The relative enrichment scores are calculated using the selected and unselected counts (see Equation 1). The two activities associated with Ras – the enzymatic activity that results in the hydrolysis of GTP, and the signaling activity that enables GTP-bound Ras to bind to effector proteins – have opposing outcomes. The enzymatic activity switches off the signaling activity, and mutations that damage the catalytic center of Ras or that increase the rate of spontaneous nucleotide exchange lead to increased signaling activity. We use the terms 'activity' and 'activation' to refer to the signaling activity of Ras. Ras is mutated frequently in cancers and some hyperproliferative developmental disorders (e.g. Noonan, Costello, and cardio-facio-cutaneous syndromes) (Li et al., 2018; Prior et al., 2020; Prior et al., 2012; Young et al., 2009). Data from cancer genomics show that most mutations occur at just three sites in Ras (Gly 12, Gly 13, and Gln 61), with the K-Ras isoform being mutated more frequently than H-Ras or N-Ras (Figure 1—figure supplement 1A, B; Tate et al., 2019). We refer to these three residues as the 'cancer hotspot' sites. In previous work, we used single-site saturation mutagenesis to assess the mutational-fitness landscape of the G-domain of H-Ras (H-Ras2-166) by employing a high-throughput bacterial two-hybrid assay (Bandaru et al., 2017). The assay couples the transcription of an antibiotic-resistance factor to the binding of Ras to the RBD of C-Raf, and reports on the signaling activity of Ras (Figure 1C). We expected mutations at Gly 12, Gly 13, and Gln 61 to be more activating than other substitutions, reflecting the frequencies of mutations observed in cancer (Figure 1—figure supplement 1A). To our surprise, the bacterial screens identified multiple signal-activating mutations in Ras, with relative enrichment scores similar to those for mutations at the three cancer hotspots. Several factors could potentially explain the difference in the mutational spectrum of Ras in cancer compared to that observed in the bacterial assay. First, the bacterial assay does not provide the complete biological context for Ras function. Ras is not membrane-localized in this system, and it is divorced from its normal complement of effector proteins. The distribution of cancer mutations reflects the mutagenic ability of specific carcinogens and the context-specific effects of Ras isoforms in different mammalian cells (Cook et al., 2021; Li et al., 2018; Prior et al., 2020). Analysis of the context-specific effects led to the proposal that there are 'sweet spots' at the intersection of these properties that are selected for in different cancers (Li et al., 2018). Nevertheless, the difference between the very narrow mutational profile of Ras in cancer and the much broader range of mutations that activate Ras in our previous deep-mutational scans is striking, and it motivated us to examine whether saturation mutagenesis could identify factors that may account for this discrepancy. To address concerns that the broad spectrum of activating mutations seen in the bacterial assay might be artifacts of this bacterial system, we carried out saturation mutagenesis of full-length H-Ras (H-Ras1-188) expressed in mammalian Ba/F3 cells (Figure 1D). The murine Ba/F3 hematopoietic cell line is dependent on the cytokine interleukin-3 (IL-3) for growth, but the IL-3 dependence can be bypassed by the expression of activated variants of tyrosine kinases, such as BCR-Abl (Daley and Baltimore, 1988; Mandanas et al., 1993; Warmuth et al., 2007). The Ba/F3 system is a robust assay for screening activating mutations in tyrosine kinases (Hoover et al., 2001; Lee and Shah, 2015; Watanabe-Smith et al., 2017). Cytokine independence can also be conferred on Ba/F3 cells by activated mutants of Ras (Awad, 2021; Hoover et al., 2001; White et al., 2016), providing the basis for our Ras saturation-mutagenesis screens. We compared the mutational-fitness landscape of full-length H-Ras1-188 in Ba/F3 cells to data obtained from bacterial saturation-mutagenesis screens done for H-Ras in the absence of GAP and GEF regulators ('unregulated Ras'), in the presence of both a GAP and a GEF ('Ras+GAP+GEF'), and in the presence of a GAP alone ('Ras+GAP'). While the first two conditions yield similar patterns of mutational sensitivity, the Ras+GAP condition leads to starkly different results. The GAP switches off Ras signaling activity, and only mutations that enable Ras to evade GAP control allow cell proliferation. The mutational profile of H-Ras in the mammalian Ba/F3 cell line closely resembles the results of the bacterial screens using H-Ras co-expressed with a GAP. These results point to the importance of GAP surveillance in controlling Ras activity in Ba/F3 cells, and also demonstrate that the bacterial assay is a reliable indicator of the effects of mutations on Ras function. Strikingly, just like in the bacterial assay, the mammalian experiments reveal additional gain-of-function mutations with similar levels of activation as mutations at the cancer hotspots. The importance of a special feature associated with the cancer hotspots emerged from the comparison of mutational profiles for longer and shorter H-Ras constructs using the bacterial assay. The shorter construct – residues 2–166, the construct typically used in crystallographic studies of H-Ras (Pai et al., 1990) – corresponds to the core G-domain of H-Ras and was used in our previous saturation-mutagenesis study. The longer construct spans residues 2–180 (H-Ras2-180), and we found that it is more stable. Our earlier study had characterized several mutations outside the hotspots that activate Ras to varying degrees, but are not prominent in cancer (Bandaru et al., 2017). We now report the analysis of protein dynamics for three of these mutations (H27G, L120A, and Y157Q) by hydrogen-deuterium exchange (HDX) measured by nuclear magnetic resonance (NMR). The mutants show increased HDX rates throughout the protein relative to wild-type Ras, consistent with destabilization. The saturation-mutagenesis data show that several infrequent cancer mutations (e.g. at Val 14 or Asp 119) are deleterious in the shorter construct and become activating in the longer Ras construct. We infer that these activating mutations have a destabilizing effect that only the longer Ras construct can accommodate, allowing the increased signaling capacity to be manifested. In contrast, the effect of mutations at the three cancer hotspots is independent of the Ras construct stability. We also present the results of saturation-mutagenesis experiments for K-Ras, which yield similar mutational patterns as for H-Ras. Our analysis shows that many activating mutations impact the thermodynamic stability of Ras, and the destabilizing effect of the mutations correlates with their low observed frequency in cancer. Results and discussion Saturation-mutagenesis of H-Ras in mammalian Ba/F3 Cells Ba/F3 cells were transduced with a retroviral library of variants of full-length human H-Ras and allowed to grow for a day in the presence of IL-3. Then, a fraction of the cells were harvested and used as the 'unselected' population. The remainder of the cells were grown for a week without IL-3 in the medium (the 'selected' population). The DNA from both cell populations was harvested and sequenced to count the occurrence of each particular variant. The effect of Ras mutations on fitness is quantified by relative enrichment scores (ΔExi), also referred to as fitness values (Equation 1; see Materials and methods): (1) ΔExi=log10[cix,selectedcix,unselected]−median(log10[Cwt,selected⊘Cwt,unselected]) The first term of Equation 1 is the logarithm of the ratio of counts (c) of observing codons representing each amino acid x at each position i in the selected and unselected samples. The second term of Equation 1 is the median of the ordered list of logarithms of the elements of the vector obtained by conducting pair-wise division, denoted ⊘, between the selected and unselected counts (Cwt,selected and Cwt,unselected, respectively) for the variants that are synonymous with the wild-type (wt) allele. A Ras variant with an enrichment score of zero propagates in the assay at the same rate as the wild-type variants. Variants with scores of ±1 propagate ten-fold faster or slower than wild-type variants, respectively. The experiment was repeated twice, once while varying residues 2–160 (the core G-domain; the remaining 29 residues were not varied), and once while varying residues 2–188. The resulting fitness values are shown in the form of a heatmap in Figure 2A, averaged over the two replicates (Figure 2—figure supplement 1A). Each entry in the matrix indicates the fitness score for substituting a particular residue in Ras with one of the 20 amino acids, with shades of red and blue indicating gain or loss-of-function relative to wild-type, respectively. Figure 2 with 2 supplements see all Download asset Open asset Mutational tolerance of Ras in mammalian Ba/F3 cells and the bacterial Ras+GAP experiment. (A) The fitness data from saturation-mutagenesis experiments are shown in the form of a matrix, where each row of the matrix represents one of the twenty natural amino acids, and each column displays a residue of the protein (Bandaru et al., 2017). Each entry in the matrix represents, in color-coded form, the relative enrichment score (ΔExi) for the corresponding variant (see Equation 1). The data are normalized using the distribution of enrichment scores of all the synonymous wild-type sequences, so the median of the distribution has a value of zero. Shades of red and blue indicate gain and loss-of-function, respectively, relative to wild-type. Green indicates variants that were not represented in the library. Stop codons are labeled as '*', and the bottom strip displays the functional effect of all amino acid substitutions at each position (⟨ΔExi⟩x) – the average taken over each column. The relative enrichment values are provided in the GitHub repository. The secondary structural elements of Ras are displayed below each matrix. The top heatmap shows the data for saturation mutagenesis of H-Ras1-188 in mammalian Ba/F3 cells. Only the enrichment scores for variants within residues 2 and 160 are displayed in the heatmap, calculated as the mean of two biological replicates. The next four heatmaps show the data for H-Ras2-166, H-Ras2-180, K-Ras2-165, and K-Ras2-173 in the bacterial Ras+GAP experiments. The enrichment values shown are the mean of two, four, three, and four biological replicates, respectively. (B) The area under the curve (AUC) of receiver operating characteristic (ROC) graphs is used to determine which of the four Ras+GAP experiments better predicts the enrichment scores of mutations in the mammalian Ba/F3 cell experiment. There are several positions in H-Ras at which multiple substitutions lead to a strong gain-of-function. These residues include the cancer hotspots (Gly 12, Gly 13, and Gln 61), as well as several other residues that are not as frequently found to be mutated in cancer (e.g. Val 14, Arg 68, Lys 117, and Asp 119). The fact that several amino acid substitutions at each of these sites lead to increased fitness suggests that the mutations disrupt inhibitory interactions. Positions at which mutations lead to a strong loss-of-function are sparse in the dataset. Residues in the hydrophobic core of the protein, for which mutations to polar residues are expected to decrease the stability of the protein, show little or no evidence for loss of fitness when mutated. For example, the sidechains of Leu 19, Leu 79, Val 81, and Val 114 pack together in the hydrophobic core of Ras. These residues tolerate substitutions by many polar residues with no apparent reduction in fitness with respect to wild-type Ras. Since only mutations that bypass the IL-3 dependence promote cell growth in the assay, and wild-type H-Ras does not promote growth (Figure 2—figure supplement 1B), a neutral mutation can barely be distinguished from a deleterious mutation. The mutational profile for Ras in Ba/F3 cells resembles that for Ras co-expressed with a GAP in the bacterial assay The two properties of the Ba/F3 dataset noted above, namely strong activation by many mutations at specific sites and a general sparsity of sites where mutations lead to loss-of-function, are reminiscent of the mutational profile for H-Ras2-166 co-expressed with a GAP (H-Ras2-166+GAP) in the bacterial assay (Bandaru et al., 2017). The GAP inactivates wild-type Ras by stimulating GTP-hydrolysis. Under these conditions, the assay is not sensitive to the effects of mutations that further reduce the activity of Ras by destabilizing the protein. Mutations that disrupt the interaction with the GAP, such as substitutions of Gly 12 or Gly 13, or that compromise the catalytic activity of Ras, such as substitutions of Gln 61, are strongly activating. We used receiver operating characteristic (ROC) curves to make a quantitative determination of which of the three different bacterial experiments [H-Ras2-166+GAP (Figure 2A), H-Ras2-166+GAP+GEF (Bandaru et al., 2017), or unregulated H-Ras2-166 (Figure 3A)] best matches the results of the Ba/F3 screen (Figure 2B). To generate a ROC curve, the fitness data for individual mutations in a particular bacterial dataset (e.g. H-Ras2-166+GAP) are used to predict the fitness of mutations in the Ba/F3 experiment. A variable threshold value of fitness is used, and for each threshold value, mutations in the bacterial dataset with a fitness value greater than that threshold are considered to predict activation in Ba/F3 data. Mutations with a fitness score greater than 1.5 times the standard deviation in the Ba/F3 dataset are considered activating (i.e. true positives). For each of the bacterial datasets, an ROC curve is generated by graphing the fraction of true positives versus the fraction of false positives at various threshold settings, and the estimated area under the curve (AUC) gives a measure of the overall prediction accuracy. For a perfect correlation between the bacterial data and the Ba/F3 data, the AUC would be 1.0. The analysis shows that the Ras+GAP dataset most accurately predicts the H-Ras1-188 in Ba/F3 cells dataset. The AUC is 0.67 for the unregulated H-Ras2-166 dataset (Figure 3—figure supplement 1) and 0.63 for H-Ras2-166+GAP+GEF (Figure 2—figure supplement 2B). For H-Ras2-166+GAP, the AUC is substantially higher at 0.84 (Figure 2B). Given that Ras is predominantly GDP-bound in vivo (Zhao et al., 2020), it is logical that the bacterial Ras+GAP experiment – where the GAP promotes a GDP-bound state – closely resembles the mammalian Ba/F3 cell experiment. Figure 3 with 1 supplement see all Download asset Open asset Mutational tolerance of Ras long and short constructs in the unregulated bacterial experiment. (A) H-Ras2-166, H-Ras2-180, K-Ras2-165, and K-Ras2-173 in the unregulated experiment. The relative enrichment values (ΔExi) shown are the mean of three, four, two, and four biological replicates, respectively. Stop codons are labeled as '*', and the bottom strip displays the functional effect of all amino acid substitutions at each position (<ΔExi>x) – the average taken over each column. (B–C) Comparison of the mutational tolerance of residues in the hydrophobic core of K-Ras in the shorter and longer constructs. The enrichment scores for the hydrophobic core residues are shown for the two K-Ras constructs in B, and mapped K-Ras in PDB ID: et al., 2015). of the C-terminal helix in Ras the correlation of bacterial mutagenesis data with the Ba/F3 dataset The Ba/F3 experiments use full-length the C-terminal that The bacterial experiments use a construct of H-Ras that spans residues 2–166, corresponding to the core domain of H-Ras (Pai et al., This construct C-terminal the where of Ras is not within the context of the bacterial assay the two-hybrid of Ras activity on the interaction between proteins Ras and to We the effects of the C-terminal of the Ras construct by conducting saturation-mutagenesis experiments with the bacterial assay using an H-Ras construct residues 2–180 (Figure on the of the K-Ras isoform et al., we that the H-Ras construct in this the C-terminal helix of H-Ras by two or more (Figure 1A). The dataset the ROC AUC score for the Ba/F3 data to compared to 0.84 for the shorter construct (Figure 2B). There are specific sites for which mutations in the bacterial experiment demonstrate a different activation profile than the H-Ras1-188 in Ba/F3 cells experiment (Figure For example, most mutations of Asp are activating in Ba/F3 cells and but these mutations are neutral or in the bacterial assay with shorter Mutations at the cancer hotspots are activating in all three (Figure and We also saturation mutagenesis of K-Ras in the presence of a GAP in bacteria using two a shorter one residues and a longer one residues (Figure 2A), corresponding to a construct used in crystallographic studies et al., 2019). The K-Ras are similar to the H-Ras (Figure The ROC AUC for the Ba/F3 data for shorter is and the AUC increases to for longer (Figure 2B). These data indicate that K-Ras to H-Ras mutational This is the review new Ras variants found in genomic studies as or on the that the of variants in the H-Ras and K-Ras are et al., 2018). We carried out saturation-mutagenesis screens in bacteria for unregulated H-Ras and K-Ras (i.e. no of GAP and for the longer and shorter constructs (Figure Ras a in the absence of the GAP and 2016), and of residues in the hydrophobic core by polar residues results in in in to the Ras+GAP experiments. The data show that the longer and K-Ras2-173 constructs are sensitive to mutations of residues in the hydrophobic core than the shorter constructs (e.g. see mutations of Figure We also screens for K-Ras in the presence of a GAP and a and the mutational landscape the same patterns for the unregulated experiments (Figure 2—figure supplement Ras mutations and construct impact stability We measured the stability of two H-Ras constructs to one corresponding to the core G-domain and one in which the C-terminal of the construct is by residues using two with Ras proteins. In the first assay, we carried out a and the by the at In the second assay, we also the using Under conditions, all the protein is and the protein et al., and et al., The fraction of folded protein can be estimated by the of the corresponding to full-length Ras on an The of the at each are then to a with the (the by used to determine The stabilities by are in with those by by the values of for the shorter and longer H-Ras constructs are and (Figure measured by the shorter and longer constructs have values of of and 1.5 (Figure indicate that the C-terminal helix H-Ras by This effect is consistent with the ability of to the protein et al., 2018). Figure Download asset Open asset stability (A) The shorter construct has an of and the longer construct has a of when measured by measured by the shorter construct has a of and the longer construct has a of 1.5 indicate that the C-terminal helix H-Ras by The were at using GDP and Ras. are two times the standard deviation of four second (B) of three cancer hotspots mutants and two gain-of-function mutants The for and mutants in the construct are 1.5 and respectively. The and variants were in the construct. The for and new replicates of wild-type are and respectively. these cancer hotspot mutations are not (C) of and has a of is more than by The experiments in were at using GDP and Ras. are the standard deviation of the estimated replicates. the assay, we measured the stability of three cancer hotspot mutants and two infrequent – or – mutants and (Figure The stabilities of the and variants of are from that of wild-type of for and for For the and variants of the values of are 1.5 and respectively. Ras by a to the effect of the C-terminal The and mutations Ras much more by Mutations at Lys and Asp as well as mutations at other sites (e.g. Val 14, Leu Lys and increase the rate of intrinsic nucleotide et al., et al., et al., 2019; et al., et al., and et al., 2019; et al., et al., two such mutations – and – have shown to nucleotide exchange by destabilization and of the et al., 2019; et al., 2019). stability and nucleotide in Ras are consistent with Ras stability being dependent on nucleotide binding and and We use the term mutations to the of mutations that from an activating

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• Ybiv from Escherichia coli K12 is a HAD phosphatase

  UNC Libraries · 2021-09-08 · 2 citations

  articleOpen access1st authorCorresponding

  The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized ybiV as a HAD phosphatase. The crystal structure of YbiV reveals a two domain protein, one with the characteristic HAD hydrolase fold, the other an inserted a/b fold. In an effort to understand the mechanism we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3) which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate ybiV may be a sugar phosphatase, which is supported by biochemical assays which measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

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• Environment and coordination of FeMo–co in the nitrogenase metallochaperone NafY

  RSC Chemical Biology · 2021-01-01 · 7 citations

  articleOpen accessCorresponding

  Broadening of NMR resonance spins used to map binding of paramagnetic FeMo–co to the nitrogenase metallocluster escort protein NafY.

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• Rotaxane Probes for the Detection of Hydrogen Peroxide by ¹²⁹Xe HyperCEST NMR Spectroscopy

  Angewandte Chemie International Edition · 2019-04-20 · 24 citations

  articleOpen access

  Abstract The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in 1 H MRI are impaired by the high abundance of protons in the body. 129 Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional 1 H MRI because of its ability to report specific chemical environments. To date, the scope of responsive 129 Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane‐based 129 Xe hyperCEST NMR contrast agent that can be turned on in response to H 2 O 2 , which is upregulated in several disease states. Added H 2 O 2 was detected by 129 Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H 2 O 2 produced by HEK 293T cells activated with tumor necrosis factor.

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• Rotaxane Probes for the Detection of Hydrogen Peroxide by ¹²⁹Xe HyperCEST NMR Spectroscopy

  Angewandte Chemie · 2019-04-20 · 5 citations

  articleOpen access

  Abstract The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in 1 H MRI are impaired by the high abundance of protons in the body. 129 Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional 1 H MRI because of its ability to report specific chemical environments. To date, the scope of responsive 129 Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane‐based 129 Xe hyperCEST NMR contrast agent that can be turned on in response to H 2 O 2 , which is upregulated in several disease states. Added H 2 O 2 was detected by 129 Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H 2 O 2 produced by HEK 293T cells activated with tumor necrosis factor.

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• Directly Functionalized Cucurbit[7]uril as a Biosensor for the Selective Detection of Protein Interactions by ¹²⁹Xe hyperCEST NMR

  Chemistry - A European Journal · 2019-03-13 · 34 citations

  articleOpen accessCorresponding

  Abstract Advancement of hyperpolarized 129 Xe MRI technology toward clinical settings demonstrates the considerable interest in this modality for diagnostic imaging. The number of contrast agents, termed biosensors, for 129 Xe MRI that respond to specific biological targets, has grown and diversified. Directly functionalized xenon‐carrying macrocycles, such as the large family of cryptophane‐based biosensors, are good for localization‐based imaging and provide contrast before and after binding events occur. Noncovalently functionalized constructs, such as cucurbituril‐ and cyclodextrin‐based biosensors, benefit from commercial availability and optimal exchange dynamics for CEST imaging. In this work, we report the first directly functionalized cucurbituril used as a xenon biosensor. Biotinylated cucurbit[7]uril (btCB7) gives rise to a 129 Xe hyperCEST response at the unusual shift of δ =28 ppm when bound to its protein target with substantial CEST contrast. We posit that the observed chemical shift is due to the deformation of btCB7 upon binding to avidin, caused by proximity to the protein surface. Conformational searches and molecular dynamics (MD) simulations support this hypothesis. This construct combines the strengths of both families of biosensors, enables a multitude of biological targets through avidin conjugation, and demonstrates the advantages of functionalized cucurbituril‐based biosensors.

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• Unconstrained peptoid tetramer exhibits a predominant conformation in aqueous solution

  Biopolymers · 2019-03-05 · 7 citations

  articleOpen access

  Abstract Conformational control in peptoids, N ‐substituted glycines, is crucial for the design and synthesis of biologically‐active compounds and atomically‐defined nanomaterials. While there are a growing number of structural studies in solution, most have been performed with conformationally‐constrained short sequences (e.g., sterically‐hindered sidechains or macrocyclization). Thus, the inherent degree of heterogeneity of unconstrained peptoids in solution remains largely unstudied. Here, we explored the folding landscape of a series of simple peptoid tetramers in aqueous solution by NMR spectroscopy. By incorporating specific 13 C‐probes into the backbone using bromoacetic acid‐2‐ 13 C as a submonomer, we developed a new technique for sequential backbone assignment of peptoids based on the 1, n ‐Adequate pulse sequence. Unexpectedly, two of the tetramers, containing an N ‐(2‐aminoethyl)glycine residue (Nae), had preferred conformations. NMR and molecular dynamics studies on one of the tetramers showed that the preferred conformer (52%) had a trans‐cis‐trans configuration about the three amide bonds. Moreover, &gt;80% of the ensemble contained a cis amide bond at the central amide. The backbone dihedral angles observed fall directly within the expected minima in the peptoid Ramachandran plot. Analysis of this compound against similar peptoid analogs suggests that the commonly used Nae monomer plays a key role in the stabilization of peptoid structure via a side‐chain‐to‐main‐chain interaction. This discovery may offer a simple, synthetically high‐yielding approach to control peptoid structure, and suggests that peptoids have strong intrinsic conformational preferences in solution. These findings should facilitate the predictive design of folded peptoid structures, and accelerate application in areas ranging from drug discovery to biomimetic nanoscience.

  PublisherOA PDFDOI

• Grassroots Efforts To Quantify and Improve the Academic Climate of an R1 STEM Department: Using Evidence-Based Discussions To Foster Community

  Journal of Chemical Education · 2019-08-19 · 23 citations

  articleOpen access

  Women and some racial and ethnic groups remain underrepresented in chemistry departments across the United States, and generally, efforts to improve representation have resulted in minimal or no improvements in the last 10 years. Here, we present the outcomes of a graduate-student-led initiative that sought to assess the issues affecting inclusivity, diversity, and wellness within the Department of Chemistry at the University of California, Berkeley. We report how the results of a department-tailored academic climate survey were used to develop a method to foster open, productive discussion among graduate students, postdoctoral researchers, and faculty. This event format led to an improved understanding of the challenges facing our community members, as well as the identification of strategies that can be used to make the Department of Chemistry more welcoming for all members. We report the success of this student-led effort to highlight the value of assessing diversity and inclusion at the department-level, as well as the benefits of using community data to stimulate productive, evidence-based discussions. Furthermore, we envision that these methods can be implemented within any research-focused academic community to promote positive cultural change.

  PublisherDOI

Recent grants

• NIH Grant S10RR015756

  NIH · $500k · 2002

• Molecular Biophysics Training Grant

  NIH · $9.6M · 1989–2023

• NIH Grant R01GM062163

  NIH · $3.1M · 2016

• NIH Grant P50GM062412

  NIH · $41.3M · 2008

• NIH Grant R01GM043129

  NIH · $1.3M · 2001

Frequent coauthors

• Alexander Pines

  University of California, Berkeley

  263 shared

• Jeffrey G. Pelton

  QB3

  126 shared

• Thomas J. Lowery

  89 shared

• John Kuriyan

  Vanderbilt University

  80 shared

• Eliseo Ruíz

  Universitat de Barcelona

  76 shared

• Matthew B. Francis

  University of California, Berkeley

  65 shared

• Peter G. Schultz

  Scripps Research Institute

  63 shared

• Seth M. Rubin

  University of California, Santa Cruz

  53 shared

Education

• Ph.D., Chemistry

  University of California Berkeley

  1979