Single-Molecule Spectroscopic Tools for Measuring Microsecond to Millisecond Dynamics of Calmodulin

Single-molecule spectroscopy has developed into a powerful tool for the study of biological systems. The ability to observe single protein changes has revealed a great deal of information about the heterogeneity of these systems. In this dissertation, single-molecule techniques have been used to investigate the effect of Ca2+ on millisecond and microsecond fluctuations of the protein calmodulin (CaM). The first part of this dissertation discusses the development and testing of a home-built, two-channel, confocal microscope system used for fluorescence correlation spectroscopy (FCS) and scanning single-molecule measurements. Secondly, the newly built system was tested by performing two-channel FCS measurements using a FRET-pair labeled synthetic polyproline peptide. Polyproline has been shown to approximate a "rigid-rod" and therefore was not expected to show any FRET fluctuations on the FCS timescale. The results from the polyproline correlations led to an investigation to develop expressions to describe the differences in the initial amplitudes of the correlations. These expressions were dependent on the presence of multiple FRET states in the solution and fits were demonstrated using both simulations and real data. Next, the dynamics of several FRET-pair labeled mutants of CaM were measured using FCS techniques. The resulting correlations were globally fit to reveal inter-lobe dynamics on the 100 microsecond timescale that were diminished upon the removal of Ca2+. Intra-lobe dynamics of the N-terminus were also investigated demonstrating an increase in the dynamics in the apo state when compared to the Ca2+ bound state. Finally, CaM was immobilized in unilamellar vesicles to probe millisecond dynamics of the CaM 34-110 mutant in the presence and absence of Ca2+. Rates of interchange between conformational substates of CaM were measured demonstrating an increase in the rates of interchange between conformations in the presence of Ca2+. This supports the view that when bound to Ca2+, CaM is in a more dynamic state leading to its ability to bind a wide variety of targets

FRET-FCS Detection of Intra-Lobe Dynamics in Calmodulin

Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca2+-signaling protein calmodulin. Intramolecular fluctuations were resolved byglobal fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius forFRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca2+ binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca2+) was nearly abolished at a high Ca2+ concentration. For the C-terminal domain the dynamic amplitude changed little with Ca2+ concentration. The Ca2+ dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca2+-binding loops

Detecting Intramolecular Dynamics and Multiple FRET States by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (greenred and red-green). We applied the method to detect intramolecular dynamics in the Ca2+ signaling protein calmodulin. Dynamics were detected on the 100-μs time scale in Ca2+-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro15-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca2+-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion

Using Empirical Phase Diagrams to Understand the Role of Intramolecular Dynamics in Immunoglobulin G Stability

Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic empirical phase diagrams. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed

Intervention for depression among palliative care patients and their families: A study protocol for evaluation of a training program for professional care staff

Background: Clinical depression is highly prevalent yet under-detected and under-treated in palliative care settings and is associated with a number of adverse medical and psychological outcomes for patients and their family members. This article presents a study protocol to evaluate a training intervention for non-physician palliative care staff to improve the recognition of depression and provide support for depressed patients and their family members. Details of the hypotheses and expected outcomes, study design, training program development and evaluation measures are described.Methods and Design: A randomised controlled trial will be implemented across two palliative care services to evaluate the &ldquo;Training program for professional carers to recognise and manage depression in palliative care settings&rdquo;. Pre-, post- and three-month follow-up data will be collected to assess: the impact of the training on the knowledge, attitudes, self-efficacy and perceived barriers of palliative care staff when working with depression; referral rates for depression; and changes to staff practices. Quantitative and qualitative methods, in the form of self-report questionnaires and interviews with staff and family members, will be used to evaluate the effectiveness of the intervention.Discussion: This study will determine the effectiveness of an intervention that aims to respond to the urgent need for innovative programs to target depression in the palliative care setting. The expected outcome of this study is the validation of an evidence-based training program to improve staff recognition and appropriate referrals for depression, as well as improve psychosocial support for depressed patients and their family members.<br /

Functional brain defects in a mouse model of a chromosomal t(1;11) translocation that disrupts DISC1 and confers increased risk of psychiatric illness

A balanced t(1;11) translocation that directly disrupts DISC1 is linked to schizophrenia and affective disorders. We previously showed that a mutant mouse, named Der1, recapitulates the effect of the translocation upon DISC1 expression. Here, RNAseq analysis of Der1 mouse brain tissue found enrichment for dysregulation of the same genes and molecular pathways as in neuron cultures generated previously from human t(1;11) translocation carriers via the induced pluripotent stem cell route. DISC1 disruption therefore apparently accounts for a substantial proportion of the effects of the t(1;11) translocation. RNAseq and pathway analysis of the mutant mouse predicts multiple Der1-induced alterations converging upon synapse function and plasticity. Synaptosome proteomics confirmed that the Der1 mutation impacts synapse composition, and electrophysiology found reduced AMPA:NMDA ratio in hippocampal neurons, indicating changed excitatory signalling. Moreover, hippocampal parvalbumin-positive interneuron density is increased, suggesting that the Der1 mutation affects inhibitory control of neuronal circuits. These phenotypes predict that neurotransmission is impacted at many levels by DISC1 disruption in human t(1;11) translocation carriers. Notably, genes implicated in schizophrenia, depression and bipolar disorder by large-scale genetic studies are enriched among the Der1-dysregulated genes, just as we previously observed for the t(1;11) translocation carrier-derived neurons. Furthermore, RNAseq analysis predicts that the Der1 mutation primarily targets a subset of cell types, pyramidal neurons and interneurons, previously shown to be vulnerable to the effects of common schizophrenia-associated genetic variants. In conclusion, DISC1 disruption by the t(1;11) translocation may contribute to the psychiatric disorders of translocation carriers through commonly affected pathways and processes in neurotransmission

Ethnic entrepreneurs and online home-based businesses: an exploratory study

This exploratory, qualitative study considers how online home-based businesses offer opportunities for ethnic entrepreneurs to ‘break out’ of traditional highly competitive and low margin sectors. Previous studies have found a positive association between ethnic minorities’ high levels of entrepreneurship and home computer use in ethnic groups. Despite these associations, previous studies have overlooked the particular opportunities offered by home-based online businesses to ethnic entrepreneurs. The study adopts mixed embeddedness as a theoretical lens to guide interviews with 22 ethnic entrepreneurs who have started online home-based businesses in the UK. We find online home-based businesses offer ethnic entrepreneurs novel opportunities to draw on their ethnic advantages and address the constraints they face. The unique affordances of this type of business allow entrepreneurs to develop the necessary IT skills by self-learning and experimentation and to sub-contract more difficult or time consuming aspects to others. The findings also show that, consistent with the theory of mixed embeddedness, whilst the entrepreneurs are influenced by social, economic and institutional forces, online businesses allow them to exert their own agency and provide opportunities to uniquely shape these forces

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands