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Andrew L Lee

Publications and source records attributed to Andrew L Lee.

16 recordsLinked to original sources
PubMed2006

Evaluation of energetic and dynamic coupling networks in a PDZ domain protein.

A number of computational and experimental studies have identified intramolecular communication "pathways" or "networks" important for transmitting allostery. Here, we have used mutagenesis and NMR relaxation methods to investigate the scope and nature of the communication networks found in the second post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain of the human protein tyrosine phosphatase 1E protein (hPTP1E) (PDZ2). It was found that most mutations do not have a significant energetic contribution to peptide ligand binding. Three mutants that showed significant changes in binding also displayed context-dependent dynamic effects. Both a mutation at a partially exposed site (H71Y) and a buried core position (I35V) had a limited response in side-chain (2)H-based dynamics when compared to wild-type PDZ2. In contrast, a change at a second core position (I20F) that had previously been shown to be part of an energetic and dynamic network, resulted in extensive changes in side-chain dynamics. This response is reminiscent to that seen previously upon peptide ligand binding. These results shed light on the nature of the PDZ2 dynamic network and suggest that position 20 in PDZ2 acts as a "hub" that is energetically and dynamically critical for transmitting changes in dynamics throughout the PDZ domain.

Amino Acid Sequence↗
PubMed2006

Cytosol has a small effect on protein backbone dynamics.

Cells are crowded with macromolecules, yet most biophysical information about proteins is obtained in dilute solution. To determine the impact of this dichotomy, we used nuclear magnetic resonance spectroscopy to measure the backbone (15)N T(1) and T(2) relaxation times and the {(1)H}-(15)N nuclear Overhauser enhancement (nOe) of uniformly (15)N-enriched apocytochrome b(5) in living Escherichia coli and in dilute solution. These data allowed us to assess the backbone dynamics of this partially folded protein in cells and in dilute solution. The two data sets were analyzed by using the model-free approach. Transfer from dilute solution to the cytosol has a quantitative effect on T(1), T(2), and nOe values. Most of the effects are attributed to an increase in the overall correlation time, caused by the increased viscosity of the cytosol compared to that of the dilute solution. Our main conclusion is that the cytosol does not alter the pattern of backbone dynamics of apocytochrome b(5). Increases in the time scale of both the picosecond and millisecond motions are observed, but the increases are less than approximately 30%.

Cell Nucleus↗
PubMed2006

Dynamic coupling and allosteric behavior in a nonallosteric protein.

Long-range intraprotein interactions give rise to many important protein behaviors. Understanding how energy is transduced through protein structures to either transmit a signal or elicit conformational changes is therefore a current challenge in structural biology. In an effort to understand such linkages, multiple V --> A mutations were made in the small globular protein eglin c. The physical responses, as mapped by NMR spin relaxation, residual dipolar couplings (RDCs), and scalar couplings, illustrate that the interior of this nonallosteric protein forms a dynamic network and that local perturbations are transmitted as dynamic and structural changes to distal sites as far as 16 A away. Two basic types of propagation responses were observed: contiguous pathways of enhanced (attenuated) dynamics with no change in structure; and dispersed (noncontiguous) changes in methyl rotation rates that appear to result from subtle deformation of backbone structure. In addition, energy transmission is found to be unidirectional. In one mutant, an allosteric conformational change of a side chain is seen in the context of a pathway of propagated changes in picosecond to nanosecond dynamics. The observation of so many long-range interactions in a small, rigid system lends experimental weight to the idea that all well-folded proteins inherently possess allosteric features [Gunasekaran et al. (2004) Proteins 57, 433-443], and that dynamics are a rich source of information for mapping and gaining mechanistic insight into communication pathways in individual proteins.

Allosteric Regulation↗
PubMed2005

Backbone and side chain dynamics of mutant calmodulin-peptide complexes.

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.

Allosteric Site↗
PubMed2005

Protein dynamics in living cells.

A protein's structure is most often used to explain its function, but function also depends on dynamics. To date, protein dynamics have been studied only in vitro under dilute solution conditions where solute concentrations are typically less than 10 g/L, yet proteins function in a crowded environment where the solute concentration can exceed 400 g/L. Does the intracellular environment affect protein dynamics? The answer will help in assessing the biological significance of the NMR-derived dynamics data collected to date. We investigated fast protein dynamics inside living Escherichia coli by using in-cell NMR. The backbone dynamics of apocytochrome b5 were quantified using {1H}-15N nuclear Overhauser effect (nOe) measurements, which characterize motions on the pico- to nanosecond time scale. The overall trend of backbone dynamics remains the same in cells. Some of the nOe values differ, but most of the differences track the increased intracellular viscosity rather than a change in dynamics. Therefore, it appears that dilute solution steady-state {1H}-15N nOe measurements provide biologically relevant information about pico- to nanosecond backbone motion in proteins.

Escherichia coli↗
PubMed2005

Role of structural plasticity in signal transduction by the cryptochrome blue-light photoreceptor.

Cryptochromes are blue-light photoreceptors that regulate a variety of responses such as growth and circadian rhythms in organisms ranging from bacteria to humans. Cryptochromes share a high level of sequence identity with the light-activated DNA repair enzyme photolyase. Photolyase uses energy from blue light to repair UV-induced photoproducts in DNA through cyclic electron transfer between the catalytic flavin adenine dinucleotide cofactor and the damaged DNA. Cryptochromes lack DNA repair activity, and their mechanism of signal transduction is not known. It is hypothesized that a light-dependent signaling state in cryptochromes is created as a result of an intramolecular redox reaction, resulting in conformational rearrangement and effector binding. Plant and animal cryptochromes possess 30-250 amino acid carboxy-terminal extensions beyond the photolyase-homology region that have been shown to mediate phototransduction. We analyzed the structures of C-terminal domains from an animal and a plant cryptochrome by computational, biophysical, and biochemical methods and found these domains to be intrinsically unstructured. We show that the photolyase-homology region interacts with the C-terminal domain, inducing stable tertiary structure in the C-terminal domain. Importantly, we demonstrate a light-dependent conformational change in the C-terminal domain of Arabidopsis Cry1. Collectively, these findings provide the first biochemical evidence for the proposed conformational rearrangement of cryptochromes upon light exposure.

Amino Acid Sequence↗
PubMed2005

Implementation and validation of a three-dimensional deformable registration algorithm for targeted prostate cancer radiotherapy.

PURPOSE: Daily prostate deformation hinders accurate calculation of dose, especially to intraprostatic targets. We implemented a three-dimensional deformable registration algorithm to aid dose tracking for targeted prostate radiotherapy. METHODS AND MATERIALS: The algorithm registers two computed tomography (CT) scans by iteratively minimizing their differences in image intensity. For validation, we measured the accuracy in registering (a) a pelvic CT set to its mathematically deformed counterpart, (b) CT scans of a deformable pelvic phantom with and without an endorectal balloon inflated, to simulate intraprostatic targets, 23 CT-opaque seeds were embedded in the prostate, and (c) two pelvic CT scans of a patient obtained on 2 separate days. RESULTS: The mean (SD) error in registering the pelvic CT set to its transformed set was 0.5 mm (1.5), with correlation coefficient improvement from 0.626 to 0.991. Using the deformable pelvic phantom, the correlation coefficient improved from 0.543 to 0.816 after registration. The mean (SD) error in tracking the intraprostatic seeds was 0.8 mm (0.5). The correlation coefficient improved from 0.610 to 0.944 after registration of the two patient CT sets. CONCLUSION: The algorithm had an accuracy of about 1 mm. It could be used for optimizing dose calculation and delivery for prostate radiotherapy.

Algorithms↗
PubMed2005

Assessing the impact of an alternative biochemical failure definition on radiation dose response for high-risk prostate cancer treated with external beam radiotherapy.

PURPOSE: The American Society for Therapeutic Radiology and Oncology (ASTRO) biochemical failure definition has recently been compared with various alternative definitions. We assessed the effect of using an alternative failure definition on the dose-response characteristics of high-risk prostate cancer treated with radiotherapy alone. METHODS AND MATERIALS: This study included 363 high-risk prostate cancer patients treated with external beam radiotherapy alone from 1987 to 1999. These patients have one or more of the following: 1992 American Joint Committee on Cancer (AJCC) digital rectal examination (DRE) stage > or = cT3, prostate-specific antigen (PSA) > 20 ng/mL, and/or biopsy Gleason score > or = 8. We previously reported the dose response based on the ASTRO definition for these patients. In this study, a biochemical failure is defined as a PSA rise > or = 2 ng/mL above the current nadir PSA (CN + 2). The failure date is defined as the time at which the event occurred (i.e., the call date). RESULTS: Using CN + 2, the tumor control probability (TCP) continues to decrease with time as opposed to reaching a plateau as with the ASTRO definition. At 5 years, TCD50 (95% CI), the dose to achieve 50% tumor control, for high-risk prostate cancer, is 70.4 (68.0-72.9) Gy using CN + 2 [ASTRO: 75.5 (70.7-80.2) Gy]. The relative slope, gamma50 (95% CI) is 1.8 (0.8-2.8) [ASTRO: 1.7 (0.7-2.7)]. Recursive partitioning again identified two subgroups: PSA < vs. > or = 13 ng/mL (ASTRO: PSA < or = vs. > 20 ng/mL). The difference in TCD50 between the two subgroups is about 20 Gy at 5 years (ASTRO: about 15 Gy at 5 years). CONCLUSION: This analysis using the CN + 2 failure definition continues to show a dose response for the high-risk group of patients. However, the dose-response characteristics differ from those estimated using the ASTRO definition. We observed that the position (TCD50) and steepness (gamma50) of the dose-response curve changed with time as long as the TCP continued to decrease. This suggests that the dose response characteristics derived from data with longer follow-up may be different from those derived with shorter follow-up using the CN + 2 or similar failure definitions which do not back-date the failure. These changes in dose-response characteristics as well as the time dependence of dose response should be noted when investigators design dose escalation trials for the high-risk prostate cancer patients.

Adenocarcinoma↗
PubMed2005

Relating side-chain mobility in proteins to rotameric transitions: insights from molecular dynamics simulations and NMR.

The dynamic aspect of proteins is fundamental to understanding protein stability and function. One of the goals of NMR studies of side-chain dynamics in proteins is to relate spin relaxation rates to discrete conformational states and the timescales of interconversion between those states. Reported here is a physical analysis of side-chain dynamics that occur on a timescale commensurate with monitoring by 2H spin relaxation within methyl groups. Motivated by observations made from tens-of-nanoseconds long MD simulations on the small protein eglin c in explicit solvent, we propose a simple molecular mechanics-based model for the motions of side-chain methyl groups. By using a Boltzmann distribution within rotamers, and by considering the transitions between different rotamer states, the model semi-quantitatively correlates the population of rotamer states with 'model-free' order parameters typically fitted from NMR relaxation experiments. Two easy-to-use, analytical expressions are given for converting S2 (axis') values (order parameter for C-CH3 bond) into side-chain rotamer populations. These predict that S2 (axis') values below 0.8 result from population of more than one rotameric state. The relations are shown to predict rotameric sampling with reasonable accuracy on the ps-ns timescale for eglin c and are validated for longer timescales on ubiquitin, for which side-chain residual dipolar coupling (RDC) data have been collected.

Carbon↗
PubMed2004

Long-range dynamic effects of point mutations propagate through side chains in the serine protease inhibitor eglin c.

Long-range interactions are fundamental to protein behaviors such as cooperativity and allostery. In an attempt to understand the role protein flexibility plays in such interactions, the distribution of local fluctuations in a globular protein was monitored in response to localized, nonelectrostatic perturbations. Two valine-to-alanine mutations were introduced into the small serine protease inhibitor eglin c, and the (15)N and (2)H NMR spin relaxation properties of these variants were analyzed in terms of the Lipari-Szabo dynamics formalism and compared to those of the wild type. Significant changes in picosecond to nanosecond dynamics were observed in side chains located as much as 13 A from the point of mutation. Additionally, those residues experiencing altered dynamics appear to form contiguous surfaces within the protein. In the case of V54A, the large-to-small mutation results in a rigidification of connected residues, even though this mutation decreases the global stability. These findings suggest that dynamic perturbations arising from single mutations may propagate away from the perturbed site through networks of interacting side chains. That this is observed in eglin c, a classically nonallosteric protein, suggests that such behavior will be observed in many, if not all, globular proteins. Differences in behavior between the two mutants suggest that dynamic responses will be context-dependent.

Alanine↗
PubMed2004

Direct demonstration of structural similarity between native and denatured eglin C.

To characterize the long-range structure that persists in the unfolded form of the 70-residue protein eglin C, residual dipolar couplings (RDCs) for HN-N and HA-CA bond vectors were measured by NMR spectroscopy for both its low pH, urea denatured state and its native state. When the data sets for the two different structural states were compared, a statistically significant correlation was found, with both sets of dipolar couplings yielding a correlation coefficient of r = 0.47 to 0.51. This finding directly demonstrates that the denatured state of eglin C has a nativelike global structure, a conclusion reached indirectly for staphylococcal nuclease by combining two different types of NMR data. A simple computer simulation showed that the degree of variation in phi and psi angles that yields the RDC correlation of r = 0.5 was inversely dependent on the statistical segment length, ranging from +/-6 to +/-30 degrees at the upper limit. Stable nativelike topologies that persist on unfolding would explain the rapid refolding kinetics displayed by many proteins and might provide a natural barrier against amyloid fibril formation.

Animals↗
PubMed2004

Ligand-dependent dynamics and intramolecular signaling in a PDZ domain.

Allosteric communication is a fundamental process that proteins use to propagate signals from one site to functionally important distal sites. Although allostery is usually associated with multimeric proteins and enzymes, "long-range" communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are small, protein-protein binding modules that can use multiple surfaces for docking diverse molecules. Furthermore, these domains have long-range energetic couplings that link the ligand-binding site to distal regions of the structure. Here, we show that allosteric behavior in a representative member of the PDZ domain family may be directly detected using side-chain methyl dynamics measurements. The changes in side-chain dynamics parameters in the second PDZ domain from the human tyrosine phosphatase 1E (hPTP1E) were determined upon binding a peptide target. Long-range dynamic effects were detected that correspond to previously observed pair-wise energetic couplings. These results provide one of the first experimental examples for the potential role of ps-ns timescale dynamics in propagating long-range signals within a protein, and reinforce the idea that dynamic fluctuations in proteins contribute to allosteric signal transduction.

Allosteric Regulation↗
PubMed2003

Increased rigidity of eglin c at acidic pH: evidence from NMR spin relaxation and MD simulations.

To gain physical insights into how proteins respond to changes in pH, the picosecond to nanosecond time scale dynamics of the small serine protease inhibitor eglin c have been studied by NMR spin relaxation experiments and MD simulations under two pH solution conditions, pH 7 and 3. Like many proteins, eglin c is destabilized by a lowering of the pH, although it retains enough stability to maintain its native conformation at pH 3. Backbone (15)N relaxation results show comparable global tumbling times (tau(m)) and model-free order parameters (S(2)) under the two pH conditions, indicating that the molecule maintains its overall molecular shape and structure at low pH, although the backbone rigidity is slightly increased ( / = 0.6%). In contrast, the side-chain methyl dynamics, as measured from (2)H relaxation experiments, show a substantial increase in rigidity at lower pH ( / = 14.8%). Molecular dynamics simulations performed at these pH states produce results consistent with NMR measurements, showing that the two methods are in qualitative agreement. Although a full accounting of the physical basis for the concurrent conformational rigidification and destabilization at low pH requires further investigation, the high level of detail in the MD simulations provides a potential molecular mechanism: the breaking of the hydrogen bond between the side chains of Asp46 and Arg53, and changes in electrostatic interactions, appear to allow the binding loop to move closer to the core part of the protein, resulting in a more compact structure at low pH. This more compact structure may be responsible for the increased level of restriction of molecular motion. As these findings show, the stability of a molecular structure is distinct from its conformational rigidity, and the two can even change in opposite directions, against naïve expectation.

Animals↗
PubMed2003

Dynamics and entropy of a calmodulin-peptide complex studied by NMR and molecular dynamics.

All-atom, explicit water molecular dynamics simulations of calcium-loaded calmodulin complexed with a peptide corresponding to the smooth muscle myosin light chain kinase target were carried out at 295 and 346 K. Amide and side chain methyl angular generalized order parameters were calculated and analyzed in the context of the protein's structure and dynamics. The agreement between amide order parameters measured by NMR and those from the simulations was found to be good, especially at the higher temperature, indicating both better convergence for the latter and excellent transferrability of the CHARMM parameters to the higher temperature. Subtle dynamical features such as helix fraying were reproduced. A large range of order parameters for the nine calmodulin methionines was observed in the NMR, and reproduced quite well in the simulations. The major determinant of the methionine order parameter was found to be the proximity to side chains of aromatic residues. An upper bound estimate of the difference in backbone entropy between loop and helical regions was extracted from the order parameters using a model of motion in an effective potential. Although loop regions are more flexible than helical regions, it was found that the entropy loss per residue upon folding was only approximately 20% less for loops than for helices. Pairwise correlated motions, which could significantly lower entropy estimates obtained from order parameter analysis alone, were found to be largely absent.

Amides↗
PubMed2002

Temperature dependence of the internal dynamics of a calmodulin-peptide complex.

The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using 15N and 2H NMR relaxation methods. NMR relaxation studies of the complex were carried out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-bearing side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains.

Animals↗