Site-specific Conformational Changes Research Articles

Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein.

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS.

Site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR

One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified 15N-Tyr labeled Ps3αHSD and its mutants, and analyzed the conformational change upon NADH binding. Using the Tyr substituted mutants, we first assigned the respective signals of four Tyr residues. In the titration experiments with NADH, the four Tyr signals changed uniquely; changes in chemical shift and signal broadening were observed. The NADH binding affinity, determined from the plots of the 1H and 15N chemical shift changes, was comparable to those reported previously. Together with the crystal structure information for Ps3αHSD in the NADH-free and -bound states, site-specific conformational changes including environmental changes could be deduced.

FTIR Analysis of Proteins and Protein-Membrane Interactions.

Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or intermolecular interactions. The sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as 13C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, the structural features of membrane pore forming proteins and peptides, and much more. The purpose of this chapter was to provide a practical guide to analyze protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques, including procedures of sample preparation, measurements, and data analysis. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, is also presented.

Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly 13C,15N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Sequential backbone resonance assignments of the E. coli dihydrofolate reductase Gly67Val mutant: folate complex.

Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(β) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35°C, and then evaluated the H(N), N(H), C(α) and C(β) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain.

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH.

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

Characterizing Induced-Conformational Changes in Intrinscially Disordered Proteins via Multi-Frequency EPR Spectroscopy

Intrinsically disordered proteins (IDPs) contain little to no secondary or tertiary structure and are often essential in biological systems. Many IDPs undergo a conformational change, where structure is induced upon binding to its target protein. Due to their very nature, structural studies of IDPs are often challenging. Here, we show how a multi-frequency approach to site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy can be utilized to characterize the mobility and conformational changes of IDPs. We have applied this method to IA3, which is a 68 residue IDP whose unstructured-to-α-helical conformational transition has been extensively characterized by various biophysical techniques. We monitored the induced conformational change in the presence of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE), at both X-, and W-band frequencies. Analyses of the X-band EPR spectral line shapes reveal that the data report on global correlation time changes consistent with a two-state model of an unstructured system and the tumbling of a rigid helix; more detailed analyzes of the X-band spectral line shapes can provide site-specific information on the residue level. Analysis of the W-band EPR spectral line shapes, however, more directly reveal site-specific structural changes. Line shape simulations of the data at both frequencies should provide further information on the site-specific conformational changes occurring in the presence of TFE and are currently underway. Using IA3 as a model system, we show multi-frequency EPR can provide insight into structural changes occurring in IDP systems that are otherwise difficult to characterize.

Probing Single-Molecule Ion Channel Dynamics by Combined Patch-Clamp Single-Molecule Fret Imaging Microscopy

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels. Such complexity makes it difficult, if not impossible, to characterize ion channel dynamics using conventional electrical recording alone since that the measurement does not specifically interrogate the associated conformational changes but rather the consequences of the conformational changes. Recently, new technology developments on single-molecule spectroscopy, and especially, the combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging have provided us the capability of probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificail lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. The function-regulating and site-specific conformational changes of ion channels are now measurable under physiological conditions in real-time, one molecule at a time. We will focus our discussion on the new development of real-time imaging of the dynamics of individual ion channels using a novel combination of single-molecule fluorescence spectroscopy and single-channel current recordings. We will then discuss a specific example of single-molecule gramicidin ion channel dynamics studied by the new approach and the future prospects.

Patch–clamp coordinated spectroscopy shows P2X 2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I(2) state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I(2) state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X(2) receptor I(2) state. However, P2X(2) receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins.

1P080 ニガウリトリプシンインヒビター変性中間体の部位特異的コンフォメーション・変化(蛋白質 C) 物生(安定性、折れたたみなど)))

1P080 ニガウリトリプシンインヒビター変性中間体の部位特異的コンフォメーション・変化(蛋白質 C) 物生(安定性、折れたたみなど)))

12] Front-face fluorescence spectroscopy of hemoglobins

This chapter focuses on the front-face fluorescence spectroscopy of hemoglobins (Hb). The intrinsic fluorescence of proteins provides a powerful spectroscopic tool to learn about dynamic protein structure. The correlation of site-specific conformational changes, as detected by spectroscopy, with the known crystal structure provides a more complete understanding of protein structural behavior. Because crystal intermolecular contacts may place a constraint on attaining the dynamic conformational states in solution, high-resolution, site-specific spectroscopy provides critical insights into protein conformation in its reactive milieu. Front-face fluorometry also has considerable applicability to the study of other macromolecules and cell organelles that have intense absorption as well as light scatter. Specific clinical applications of front-face fluorometry are well proven as highly sensitive assay. This chapter concludes that the intrinsic fluorescence of Hb is useful as a probe to assay dynamic, site-specific conformational changes in normal and mutant Hb.

DNase I hypersensitive sites along the XY bivalent at meiosis in man include the XpYp pairing region.

The DNase I nick translation technique has been applied to human meiotic chromosomes in situ. At metaphase I, distinct hot spots of autoradiographic labelling occur at three positions along the XY bivalent; over the Xpter and Ypter pairing tips, over Xq and Yq terminal/telomeric segments, and at a site just below the centromere in Xq. The latter might correspond to the postulated human inactivation centre. Compared with somatic chromosomes, human meiotic bivalents in general exhibit a greater accessibility to DNase I. Site-specific conformational changes in the DNA between somatic and germ line cells could be a necessary prerequisite for crossing-over.