Biomedical subjects

Michael W Clarkson

Publications and source records attributed to Michael W Clarkson.

4 recordsLinked to original sources

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

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↗

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↗

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↗