Compact Conformational State Research Articles

Understanding familial Alzheimer's disease: The fit‐stay‐trim mechanism of γ‐secretase

AbstractUnderstanding Alzheimer's disease is a central challenge of the 21st century, as the disease affects tens of millions of people and kills a million people each year, with current drugs having modest effect. This article reviews how computational science integrating new cryo‐electron microscopy structures and biochemical and clinical data has led to a causative model of familial Alzheimer's disease (fAD). The model's basis is open and compact conformational states of the membrane protease γ‐secretase, controlled by transmembrane helix “fingers” that hold the substrate either tightly or loosely. The two states are in thermal equilibrium and lead to different amounts of long and short Aβ peptides, explaining the much‐debated Aβ42/Aβ40 ratio. Pathogenic mutations shift the equilibrium toward the open state by reducing the stability and hydrophobic packing of the enzyme‐substrate complex, which increases toxic Aβ42 and other longer peptide forms compared with Aβ40. In contrast, drugs that selectively target longer, pathogenic Aβ peptides should preferentially stabilize the compact state to reverse this tendency. The model may explain how inherited mutations cause fAD and provides a molecular roadmap for the development of γ‐secretase modulators, one of the most promising causative treatment strategies in current Alzheimer research. In summary, we showcase the power of modern multiscale computational science in integrating biochemical, protein‐structural, and clinical data to elucidate complex disease mechanisms.This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Data Science > Chemoinformatics

Necessity of High Physical Resolution in the Development of Flexible Coarse-Grained Protein Models

The Martini force field is a one of the most widely used coarse-grained (CG) model. An important strength of the Martini force field is that it includes explicit, microscopic representation of solvent, which allows proper description of the context-dependence of protein-protein interactions. However, in the Martini force field the secondary structures of the protein are fixed, which makes it is unsuitable for simulating dynamic processes such as protein folding and membrane insertion. In this work, we examine the possibility of developing a flexible protein model within the Martini CG framework, such as by supplementing the force field with Go-like potentials. It was found the current Martini CG representation of proteins was insufficient to support a flexible model. The model does not provide a sufficient resolution to properly describe the volume and packing of protein backbone and sidechains. The interior voids resulting from improper packing would lead to excessive structural collapse in absence of structural restraints. Along this line, we further examined the PACE model, where an atomistic protein model is deployed in the Martini solvent. The results suggest that, while atomistic protein representation does dramatically improve the ability to describe specific protein-protein interactions, the low physical resolution of the Martini water molecules does not allow sufficient discrimination of various open and compact conformational states. As a result, PACE is not capable of describing context dependent protein structure transition, such as helical transition induced by membrane absorption and/or insertion. In conclusion, high physical resolution is likely necessary for developing flexible protein models and the coarse-graining needs to focus on exploiting possible simplification of interaction potentials.

A Compact Intermediate State of Calmodulin in the Process of Target Binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.

Single-Molecule Dynamics of Conformational Interchange in Calmodulin

The recognition and binding of target binding domains by calmodulin (CaM) require global protein conformational changes. We describe single-molecule measurements of conformational interchange in CaM. CaM labeled with a FRET pair (Alexa Fluor 488 and Texas Red) was trapped in lipid vesicles tethered to the surface of a cover slip. Conformational transitions were observed on the time scale of 2 to 11 ms. The probability of a transition to a compact conformation was significantly lower at low than at high Ca2+ concentration, revealing more frequent transitions to a compact conformational state for CaM with bound Ca2+. These results show that CaM undergoes functional conformational dynamics even in the absence of target enzyme. Conformational searching may permit CaM to readily adopt a binding geometry upon encountering a target binding domain.FRET trajectories were also analyzed by a novel iterative Bayesian propagator to determine the probability distribution of FRET efficiencies as a function of time. We show that this analysis outperforms conventional running averages to resolve conformational jumps and conformational distributions. The figure shows a CaM FRET efficiency probability distribution (color scale). The red line shows the running average. The time axis is in milliseconds.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A shorter peptide model from staphylococcal nuclease for the folding–unfolding equilibrium of a β-hairpin shows that unfolded state has significant contribution from compact conformational states

It is important to understand the conformational features of the unfolded state in equilibrium with folded state under physiological conditions. In this paper, we consider a short peptide model LMYKGQPM from staphylococcal nuclease to model the conformational equilibrium between a hairpin conformation and its unfolded state using molecular dynamics simulation under NVT conditions at 300K using GROMOS96 force field. The free energy landscape has overall funnel-like shape with hairpin conformations sampling the minima. The "unfolded" state has a higher free energy of approximately 12kJ/mol with respect to native hairpin minimum and occupies a plateau region. We find that the unfolded state has significant contributions from compact conformations. Many of these conformations have hairpin-like topology. Further, these compact conformational forms are stabilized by hydrophobic interactions. Conversion between native and non-native hairpins occurs via unfolded states. Frequent conversions between folded and unfolded hairpins are observed with single exponential kinetics. We compare our results with the emerging picture of unfolded state from both experimental and theoretical studies.

Alkaline-induced unfolding and salt-induced folding of pig heart lactate dehydrogenase under high pH conditions

The alkaline-induced unfolding and the salt-induced folding of pig heart lactate dehydrogenase under high pH conditions have been followed by fluorescence emission spectra and circular dichroism spectra. The results for alkaline-induced denaturation of lactate dehydrogenase show that at low ionic strength, increasing the pH value increased the extent of unfolding of the enzyme to the maximum ultimate unfolded conformation at about pH 13.0. At pH 12.5, although the enzyme was completely inactivated, most of the ordered structure was retained. Even at pH 13.5, the apparently fully unfolded enzyme still retained some ordered secondary structure. Kinetic analysis showed that at high pH, the inactivation rate constants of the enzyme are an order of magnitude faster than the unfolding rate constants at least. The above results are in accord with the suggestion by Tsou (Trends Biochem Sci 1986;11:427–429 and Science 1993;262:380–381) that the active site is usually more flexible than the enzyme molecule. At pH 13.0, adding salt to the solution caused the relatively unfolded state of the denatured enzyme to change into a compact conformational state by hydrophobic collapsing. The folded state induced by the salt bound ANS strongly, indicating the existence of an increased hydrophobic surface. The above results suggest that the salt-induced folded state at high pH may be the folded intermediate which exists in the general protein folding and that the large residual ordered secondary structure might become folded during the salt-induced folding.

Salt-induced folding of alkaline denatured creatine kinase under high pH conditions.

The conformational changes of creatine kinase during alkaline unfolding and salt-induced folding at high pH have been followed by fluorescence emission and circular dichroism spectra. The results obtained show that at low ionic strength, with increasing pH value, creatine kinase denatured gradually to reach the ultimate unfolded conformation in the vicinity of pH 12.7. With the increase of pH from 9.0 to 12.7, the fluorescence emission maximum red shifted from 337 to 355 nm, indicating complete exposure of the buried tryptophan residues to the solvent. The far-UV CD spectra show that even at pH 12.7, the apparently fully denatured enzyme retains a great part of ordered secondary structure. At pH 12.7 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing. Folded state induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface. The above results suggest that the salt-induced folded state at high pH may be the folded intermediate which exists in the general protein folding, and the larger residual ordered secondary structure might become folded being point on the salt-induced folding.

Alkaline unfolding and salt-induced folding of yeast alcohol dehydrogenase under high pH conditions.

The conformational changes of yeast alcohol dehydrogenase during unfolding at alkaline pH have been followed by fluorescence emission and circular dichroism spectra. A result of comparison of inactivation and conformational changes shows that much lower values of alkaline pH are required to bring about inactivation than significant conformational change of the enzyme molecule. At pH 9.5, although the enzyme has been completely inactivated, no marked conformational changes can be observed. Even at pH 12, the apparently fully unfolded enzyme retains some ordered secondary structure. After removal of Zn2+ from the enzyme molecule, the conformational stability decreased. At pH 12 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing. Folded states induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface. More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-enzyme was lower than that of holo-enzyme. The above results suggest that the zinc ion plays an important role in helping the folding of YADH and in stabilizing its native conformation.

Sec-dependent preprotein translocation in bacteria.

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multisubunit protein complex termed translocase, which consists of the integral membrane heterotrimer Sec YEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with Sec YEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles.

Alkaline unfolding and salt-induced folding of aminoacylase at high pH.

The conformational changes of aminoacylase during unfolding at alkaline pH have been followed by fluorescence emission, circular dichroism (CD) and ultraviolet difference spectra. The results of comparison of inactivation and conformation show that much lower values of alkaline pH are required to bring about inactivation than significant conformational change of the enzyme molecule. At pH above 12, although the enzyme has been inactivated, the apparently fully unfolded enzyme retains some ordered secondary structure. At pH 12 by adding KCl, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing, but no new secondary structure is formed. On decreasing the pH from pH 12 to approximate neutrality, the unfolded enzyme also undertakes the similar conformational transition. It can be suggested that hydrophobic collapsed intermediate may be a general intermediate conformational state from alkaline unfolded state to native state.

Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: Its allotypes differ in conformation

Human placental microvillous alkaline phosphatase (M-PLAP) was extracted from microvilli either by butanol extraction or subtilisin proteolysis. The data indicate that subtilisin cleavage of PLAP removes a membrane-binding domain of approximately 2000 molecular weight, leaving the catalytic site intact and the protein in solution. Sequencing studies on the N-terminal 13 amino acids of both the subtilisin-cleaved and uncleaved forms of M-PLAP indicate that the enzyme is anchored to the plasma membranes by its carboxy-terminus. The N-terminal 13 amino acids of A-PLAP were the same as those of M-PLAP. Trypsin solubilization failed to release M-PLAP from these membranes and it appears to cleave a portion of molecular weight of about 9K from the amino terminus, leaving an enzymatically active portion of PLAP associated with the membrane. On SDS gels, subtilisin-cleaved M-PLAP showed an apparent dimeric molecular size larger than that of the original uncleaved enzyme, presumably due to the generation of a less compact conformational state. On starch gels, cleaved M-PLAP showed a single zone of enzyme activity with a mobility sightly greater than that of A-PLAP, which did not require the presence of Triton X-100 to enter the gel. Variations in the apparent molecular sizes of the different allelic forms of PLAP were also observed.

Unfolding and refolding of Staphylococcus aureus penicillinase by urea-gradient electrophoresis

The unfolding and refolding of Staphylococcus aureus penicillinase have been followed by urea-gradient electrophoresis. Unfolding of the native state proceeds by an all-or-none transition to fully unfolded protein, with no detectable accumulation of partially unfolded states. In contrast, refolding is complex and proceeds by very rapid, reversible formation of a partially folded state, H, which had been detected and characterized previously, as it is the most stable conformation at intermediate denaturant concentrations. At very low urea concentrations, a more compact conformational state was observed as a transient intermediate in refolding. There was little kinetic heterogeneity of the unfolded protein, as is normally observed with proteins containing proline residues.

On the Mechanisms of pH-dependent Hydrogen Exchange of Bovine Plasma Albumin in the Range of pH 5 to 8.5

Hydrogen exchange rates of bovine plasma albumin vary over the range of pH 5 to 8.5. As the pH is raised, the number of very slowly exchanging hydrogens decreases and the number of rapidly exchanging hydrogens steadily increases. The rate of exchange of hydrogens of the very slowly exchanging core at pH 5 was studied at pH values from pH 5.2 to pH 8.5. The results obtained suggest that this protein is highly motile; that is, it fluctuates rapidly at pH 7 and above between states in which exchangeable hydrogens are accessible to bulk solvent and ones in which they are not accessible. In the range of pH 5 to 6.5, no segments of the core appear to be highly motile but compact segments may become highly motile as the pH is raised through this range. The alternative possibility, that changes in exchange behavior at pH 5.2 to 6.5 are the results of changes of the local environment of individual exchanging groups, cannot be excluded. The effects of sodium dodecyl sulfate, glycerol, and changing ionic strength were also studied at pH 5 and 7.7. The results were consistent with three possible explanations: (a) that these agents stabilize compact conformational states of segments of bovine plasma albumin; (b) that they reduce segmental conformational motility;; or (c) that they alter the local environment of individual exchanging groups and thus influence the rate dependencies of exchange of these units. Discrimination between these three mechanisms was not deemed possible on the basis of our experimental data.