The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.     

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43. Supported by the National Natural Science Foundation of China (Grant No. 39890390)     

Thermal fluctuation spectroscopy of DNA thermal denaturation

We have developed the technique of thermal fluctuation spectroscopy to measure the thermal fluctuations in a system. This technique is particularly useful to study the denaturation dynamics of biomolecules like DNA. Here we present a study of the thermal fluctuations during the thermal denaturation (or melting) of double-stranded DNA. We find that the thermal denaturation of heteropolymeric DNA is accompanied by large, non-Gaussian thermal fluctuations. The thermal fluctuations show a two-peak structure as a function of temperature. Calculations of enthalpy exchanged show that the first peak comes from the denaturation of AT rich regions and the second peak from denaturation of GC rich regions. The large fluctuations are almost absent in homopolymeric DNA. We suggest that bubble formation and cooperative opening and closing dynamics of basepairs causes the additional fluctuation at the first peak and a large cooperative transition from a partially molten DNA to a completely denatured state causes the additional fluctuation at the second peak.     

Structural characteristics of extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 core antenna complexes CP43 and CP47

The structural characteristics of the extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 (PS2) core antenna complexes CP43 and CP47 were investigated using fluorescence emission and circular dichroism (CD) spectra. The extra-membrane domains of CP43 and CP47 possessed a certain degree of secondary and tertiary structure and not a complete random coil conformation. The tertiary structure and the chlorophyll (Chl) a microenvironment of CP47 were more sensitive to guanidine hydrochloride (GuHCl) than that of CP43. Changes in energy transfer from β-carotene to Chl a corresponded well to changes in the tertiary structure while their correlation with changes in the secondary structure was rather poor. Unlike most of water-soluble proteins, both CP43 and CP47 are partly resistant to denaturation induced by guanidine hydrochloride (GuHCl); the denaturation of CP43 or CP47 is not a two-state process. Those features most probably reflect their character as intrinsic membrane proteins.     

Application of ATR‐FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR‐FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR‐FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α–helix to β‐sheet or intermolecular β‐sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR‐FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 574–584, 2015.     

Interaction of mitoxantrone, as an anticancer drug, with chromatin proteins, core histones and H1, in solution

In the present study, for the first time we have investigated the interaction of anticancer drug mitoxantrone with histone H1 and core histone proteins in solution using fluorescence, UV/Vis, CD spectroscopy and thermal denaturation techniques. The results showed that mitoxantrone reduced the absorbencies of H1 and core histone proteins at 210 nm (hypochromicity) and fluorescence emission intensity was decreased in a dose dependent. Binding of mitoxantrone changed secondary structures of the proteins as circular dichroism analysis confirmed it. Also, mitoxantrone increased the melting temperature of core histones at the final step of denaturation. The results suggest higher affinity of mitoxantrone to histone H1 compared to core histones providing histone proteins as a new target for mitoxantrone action at the chromatin level.     

The structure of plant vacuolar membranes according to infrared spectroscopy

The structure of the vacuolar membrane (tonoplast) was studied in red beet roots by IR spectroscopy. The vacuolar membrane was shown to be composed of highly ordered lipids which form regions of free liquid lipid bilayer loosely bound to integral proteins. The prevalence of polar lipids in the tonoplast is responsible for the high elasticity and fluidity of the membrane. The presence of alpha-tocopherol in the tonoplast membrane accounts for a high antioxidant activity of the membrane. Integral proteins are immersed into the liquid matrix of the lipid bilayer to a different extent. Examination of the temperature effect on the kinetics of the hydrogen-deuterium exchange in integral membrane proteins showed that the efficient energy of the hydrogen exchange activation was 24 +/- 4 kcal/mol at 19-40 degrees C and increased to 54 kcal/mol at 40-50 degrees C because of the thermal denaturation of proteins. The secondary structure of integral membrane proteins is characterized by a high content of alpha-helices (53%) which decreased to 8% after the extraction of lipids.     

High-resolution waveguide THz spectroscopy of biological molecules

Low-frequency vibrational modes of biological molecules consist of intramolecular modes, which are dependent on the molecule as a whole, as well as intermolecular modes, which arise from hydrogen-bonding interactions and van der Waals forces. Vibrational modes thus contain important information about conformation dynamics of biological molecules, and can also be used for identification purposes. However, conventional Fourier transform infrared spectroscopy and terahertz time-domain spectroscopy (THz-TDS) often result in broad, overlapping features that are difficult to distinguish. The technique of waveguide THz-TDS has been recently developed, resulting in sharper features. For this technique, an ordered polycrystalline film of the molecule is formed on a metal sample plate. This plate is incorporated into a metal parallel-plate waveguide and probed via waveguide THz-TDS. The planar order of the film reduces the inhomogeneous broadening, and cooling of the samples to 77K reduces the homogenous broadening. This combination results in the line-narrowing of THz vibrational modes, in some cases to an unprecedented degree. Here, this technique has been demonstrated with seven small biological molecules, thymine, deoxycytidine, adenosine, D-glucose, tryptophan, glycine, and L-alanine. The successful demonstration of this technique shows the possibilities and promise for future studies of internal vibrational modes of large biological molecules.     

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.     

Structural study of the C2 domains of the classical PKC isoenzymes using infrared spectroscopy and two-dimensional infrared correlation spectroscopy

The secondary structure of the C2 domains of the classical PKC isoenzymes, alpha, betaII, and gamma, has been studied using infrared spectroscopy. Ca(2+) and phospholipids were used as protein ligands to study their differential effects on the isoenzymes and their influence on thermal protein denaturation. Whereas the structures of the three isoenzymes were similar in the absence of Ca(2+) and phospholipids at 25 degrees C, some differences were found upon heating in their presence, the C2 domain of the gamma-isoenzyme being better preserved from thermal denaturation than the domain from the alpha-isoenzyme and this, in turn, being better than that from the beta-isoenzyme. A two-dimensional correlation study of the denaturation of the three domains also showed differences between them. Synchronous 2D-IR correlation showed changes (increased aggregation of denaturated protein) occurring at 1616-19 cm(-1), and this was found in the three isoenzymes. On the other hand, the asynchronous 2D-IR correlation study of the domains in the absence of Ca(2+) showed that, in all cases, the aggregation of denaturated protein increased after changes in other structural components, an increase perhaps related with the hard-core role of the beta-sandwich in these proteins. The differences observed between the three C2 domains may be related with their physiological specialization and occurrence in different cell compartments and in different cells.     

Vibrational circular dichroism spectra of protein films: thermal denaturation of bovine serum albumin

Vibrational circular dichroism (VCD) spectroscopy has been used for the first time to investigate the thermal denaturation of proteins in H(2)O solutions. Films prepared from heated aqueous solutions were used for these investigations. A well-known alpha-helical protein, bovine serum albumin (BSA), is used for this first study. Both VCD and infrared absorption results obtained for BSA films indicate that the heat treatment of BSA induces significant amounts of beta-sheet structure and that the denaturation process is irreversible. To verify the irreversible nature of thermal denaturation, optical rotation was also measured as a function of temperature in both heating and cooling cycles. These results also indicate that thermal denaturation of BSA in solution is irreversible. This study establishes the usefulness of films for VCD investigations and offers new avenues for VCD studies on biologically important systems.     

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.     

The periplasmic E. coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

Heat-induced changes in the photochemical centres and the protein secondary structures of photosystem II studied by variable fluorescence and difference FT-IR spectroscopy

Variable fluorescence (Fv), i.e., Fv = Fm-Fo where Fo is the minimal fluorescence and Fm the maximum fluorescence, and difference Fourier transform infrared (FT-IR) spectroscopy were used to study the effect of heat stress in the 25-55 degrees C range on photosystem II (PSII) structure and function. First, the Fv intensity reflects accurately the changes in the number of open photochemical centers in PSII. Secondly, the use of Fv in combination with FT-IR spectroscopy can disclose structure-function correlations in the heat inactivation of the PSII complex. Analysis of the midpoint temperatures of thermal denaturation, i.e., 50% inactivation, reported so far in investigations of the thylakoid membrane components has revealed that most of the thermal transitions attributed to PSII are in the 39-46 degrees C range. In this work, it is shown specifically that the midpoint temperature of PSII inactivation is at about 40 degrees C. Moreover, it was clearly demonstrated that the heat-induced changes above 40 degrees C are the result of a marked decrease in the number of open photochemical centers in PSII. It was also seen that above this same temperature the loss of photochemical centers has its structural counterpart in overall modifications of the secondary structures of the PSII proteins resulting from the decrease in the alpha-helix content concomitant with the increase in extended chain (beta-strand) conformations. In brief, a novel finding reported here is that the number of open photochemical centers in PSII is dependent on a dynamic equilibrium between the contents of the PSII proteins in alpha-helix and extended chains (beta-strands), but not in beta-sheets and beta-turn structures except for the antiparallel-beta-sheet conformations. This therefore associates the thermal inactivation of the photochemical centers in photosystem II with distinct conformational changes in the proteins of the PSII supramolecular complex. In the particular context of the present study, these findings constitute a significant contribution to the investigation of structure-function correlations in the photosynthetic membrane. In a broader context, this information might be essential for the comprehension of the molecular arrangements or local structure order that are involved directly or indirectly in biological catalysis.     

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.     

Molecular basis of the heat denaturation of photosystem II

The thermal denaturation of the photosystem II (PSII) membrane protein complex is investigated by assigning the endothermic transitions observed by differential scanning calorimetry (DSC) to the denaturation of particular proteins of the PSII complex. In a prior DSC study of PSII membranes [Thompson, L. K., Sturtevant, J. M., & Brudvig, G. W. (1986) Biochemistry 25, 6161], five DSC peaks were observed in the 30-70 degrees C temperature range (A1, A2, B, C, and D). The A2 peak was assigned to denaturation of a component essential for water oxidation and the B peak to denaturation of a component critical to the remainder of the electron-transport chain. We have now extended these studies with thermal gel analysis and electron paramagnetic resonance (EPR) measurements. Thermal gel analysis, a technique which relies on a change in the solubility properties of a membrane protein upon denaturation, has been used to determine the temperatures of denaturation of all of the major membrane proteins of the PSII complex. EPR experiments have been used to monitor chlorophyll photooxidation and the stability of TyrD+. Peaks B, C, and D in the DSC denaturation profile are each assigned to the denaturation of several proteins, which provides information on the organization of the PSII complex into structural and functional units. Peak B corresponds to the denaturation of peripheral core proteins and closely associated antenna proteins, peak C to the PSII core, and peak D to the loosely associated antenna proteins. No membrane protein is observed to denature during the A2 peak. The A2 peak is altered by the presence of catalase, superoxide dismutase, low chloride, and high pH. These results suggest that the abnormally sharp A2 peak occurs when the highly oxidizing, sequestered Mn complex (the active site in water oxidation) becomes accessible to the aqueous phase, at elevated temperatures. We propose a mechanism for the reaction of the Mn complex with hydroxide ions, which involves peroxide or superoxide and results in the reduction and release of Mn. The proposed model provides insight into the well-known instability of the Mn complex and the role of chloride in stabilizing the complex. This may enable the future development of purification procedures and may explain the sensitivity of the water-oxidizing apparatus of PSII to heat denaturation.     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

Solid-state NMR spectroscopy applied to membrane proteins

One major remaining problem in structural biology is to elucidate the structure and mechanism of function of membrane proteins. On the basis of preliminary information from genome projects, it is now estimated that up to 50,000 different membrane proteins may exist in the human being and that virtually every life process proceeds, sooner or later, through a membrane protein. Solid-state NMR spectroscopy in high magnetic field is rapidly developing into a widely applicable tool to describe the structure and help understand the mechanism of function of a membrane protein. Recent work in applied solid-state NMR spectroscopy crosses the boundary between the biological and the physical sciences, and aims at increasing the predictive range of this biophysical method.