CONFORMATIONAL DYNAMICS AND ALLOSTERY IN SIGNALING PROTEINS: MONITORING TRANSIENT STATES BY STRUCTURAL MASS SPECTROMETRY

Ph.DDOCTOR OF PHILOSOPH

Use of Tetra-ammonium Tetrakis(4-Sulphonato)Phenyl Porphyrin for Pseudomonas and Bacillus Cell Imaging

The use of tetraammonium tetrakis(4-sulphonato)phenyl porphyrin (TPPS), a water-soluble anionic compound, as a stain to analyse bacterial cells using fluorescent microscopy was investigated. TPPS was effectively used to analyse two different bacteria: Pseudomonas aeruginosa and Bacillus cereus. The variation in brightness with varying concentrations of TPPS was studied. The patterns of variations for these bacteria were found to be the same, but with consistently higher brightness for Bacillus cereus

A nexus of intrinsic dynamics underlies translocase priming

The cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We show that priming exploits a nexus of intrinsic dynamics in SecA. Using atomistic simulations, smFRET, and HDX-MS, we reveal multiple dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are functionally important and conserved. Central to the nexus is a slender stem through which rotation of the preprotein clamp of SecA is biased by ATPase domain motions between open and closed clamping states. An H-bonded framework covering most of SecA enables multi-tier dynamics and conformational alterations with minimal energy input. As a result, cognate ligands select preexisting conformations and alter local dynamics to regulate catalytic activity and clamp motions. These events prime the translocase for high-affinity reception of non-folded preprotein clients. Dynamics nexuses are likely universal and essential in multi-liganded proteins.</p

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly “catch and release” trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted

Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design - Fig 2

<p><b>(A)</b> A modified mirror plot of relative deuterium uptake values (y-axis) for every pepsin digest peptide analyzed, listed from the N to C terminus (x-axis) of Hsp90 for each time point of deuterium exchange. Reporter Regions were determined according to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004840#pcbi.1004840.e001" target="_blank">Eq 1</a> and boxed in teal. <b>(B, C)</b> Regions showing high dynamics were mapped onto the surface representation of Hsp90 (PDB Id: 4EGK) in blue bound to Radicicol (green sticks).</p

Moderated Test Statistics to Detect Differential Deuteration in Hydrogen/Deuterium Exchange Mass Spectrometry Experiments

With differential hydrogen/deuterium exchange, differences in the structure and dynamics of protein states can be studied. Detecting statistically significant differentially deuterated peptides is crucial to draw meaningful conclusions about the distinct conformations and dynamics of the protein under study. Here, we introduced a linear model in combination with an empirical Bayes approach to detect differentially deuterated peptides. Using a linear model allows one to test for differences in deuteration between two (two-sample t-test) or more groups (F-statistic), while potentially controlling for the effects of other variables that are not of interest. The empirical Bayes approach improves the estimation of deuteration-level variances, especially in experiments with a low number of replicates. As a consequence, the two sample t-tests and the F-statistic become moderated, resulting in a lower number of false positive and false negative findings. Furthermore, we introduce a thresholded-moderated t-statistic to test if the observed deuteration differences are larger than a specified, biologically relevant difference. Finally, we underline the importance of having a sufficient number of replicates, and the effect of the number of replicates on the power of the statistical significance tests. The R-code for the proposed moderated test statistics is available upon request

Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase

Bacterial secretory preproteins are translocated across the inner membrane post-translationally by the SecYEG-SecA translocase. Mature domain features and signal peptides maintain preproteins in kinetically trapped, largely soluble, folding intermediates. Some aggregation-prone preproteins require chaperones, like trigger factor (TF) and SecB, for solubility and/or targeting. TF antagonizes the contribution of SecB to secretion by an unknown molecular mechanism. We reconstituted this interaction in vitro and studied targeting and secretion of the model preprotein pro-OmpA. TF and SecB display distinct, unsuspected roles in secretion. Tightly associating TF:pro-OmpA targets the translocase at SecA, but TF prevents pro-OmpA secretion. In solution, SecB binds TF:pro-OmpA with high affinity. At the membrane, when bound to the SecA C-tail, SecB increases TF and TF:pro-OmpA affinities for the translocase and allows pro-OmpA to resume translocation. Our data reveal that TF, a main cytoplasmic folding pathway chaperone, is also a bona fide post-translational secretory chaperone that directly interacts with both SecB and the translocase to mediate regulated protein secretion. Thus, TF links the cytoplasmic folding and secretion chaperone networks.status: publishe

Fragments 1 and 2 differ in the nature of the allosteric effect in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (inferred from difference in mass in Daltons (Da) (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to the apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to establish allosteric regions (red boxed). Fragment <b>2</b> does not show any changes in region A4, similar to 17-AAG, while fragment <b>1</b> shows differences, similar to Radicicol. In addition, fragment <b>1</b> shows an allosteric response at the regions A5 (residues 201–213 shown in orange box), which is not observed in the other three ligands. Time points are colored according to key. <b>(B,C)</b> The identified orthosteric (blue) and allosteric regions (red) for fragments are mapped on to the structure of Hsp90 in blue. <b>(C)</b> The allosteric site A5 in Hsp90, which is observed only fragment <b>2</b> is highlighted in orange. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p