Structural characterization of MG and pre-MG states of proteins by MD simulations,NMR, and other techniques

Almost all proteins fold via a number of partially structured intermediates such as molten globule (MG) and pre-molten globule states. Understanding the structure of these intermediates at atomic level is often a challenge, as these states are observed under extreme conditions of pH, temperature, and chemical denaturants. Furthermore, several other processes such as chemical modification, site-directed mutagenesis (or point mutation), and cleavage of covalent bond of natural proteins often lead to MG like partially unfolded conformation. However, the dynamic nature of proteins in these states makes them unsuitable for most structure determination at atomic level. Intermediate states studied so far have been characterized mostly by circular dichroism, fluorescence, viscosity, dynamic light scattering measurements, dye binding, infrared techniques, molecular dynamics simulations, etc. There is a limited amount of structural data available on these intermediate states by nuclear magnetic resonance (NMR) and hence there is a need to characterize these states at the molecular level. In this review, we present characterization of equilibrium intermediates by biophysical techniques with special reference to NMR.     

A novel mechanism for the modulation of the Ras-effector interaction by small molecules

When proteins require different conformations for their biological function, all these functional states have to coexist simultaneously in solution. However, the corresponding Gibbs free energy differences are usually rather high and thus the conformation with lowest energy predominates in solution whereas the populations of the states with higher energy (excited states) are very small. A stabilization of these excited states can be used as a novel principle to influence the activity of proteins by small molecules. For a proof of this principle, we selected the Ras protein that was shown by (31)P NMR spectroscopy to exist in solution in at least two different conformational states in its GTP form. One of these states shows a drastically reduced affinity to effectors. With Zn(2+)-cyclen we found a small molecule which selectively stabilizes the weak-binding state. It may serve as lead compound for the development of a new type of Ras-inhibitors.     

The small heat-shock chaperone protein, alpha-crystallin, does not recognize stable molten globule states of cytosolic proteins

The small heat-shock protein (sHsp), alpha-crystallin, acts as a molecular chaperone by interacting with destabilized 'substrate' proteins to prevent their precipitation from solution under conditions of stress. alpha-Crystallin and all sHsps are intracellular proteins. Similarly to other chaperones, the 'substrate' protein is in an intermediately folded, partly structured molten globule state when it interacts and complexes with alpha-crystallin. In this study, stable molten globule states of the cytosolic proteins, gamma-crystallin and myoglobin, have been prepared. Within the lens, gamma-crystallin naturally interacts with alpha-crystallin and myoglobin and alpha-crystallin are present together in muscle tissue. The molten globule states of gamma-crystallin and myoglobin were prepared by reacting gamma-crystallin with glucose 6-phosphate and by removing the haem group of myoglobin. Following spectroscopic characterisation of these modified proteins, their interaction with alpha-crystallin was examined by a variety of spectroscopic and protein chemical techniques. In both cases, there was no interaction with alpha-crystallin that led to complexation. It is concluded that alpha-crystallin does not recognise stable molten globule states of cytosolic 'substrate' proteins and only interacts with molten globule states of proteins that are on the irreversible pathway towards an aggregated and precipitated form.     

Slips,leaks and channels in glutamate transporters

Glutamate transporters are unusual proteins in that they can function as both a transporter and a chloride channel. With the recent determination of the crystal structure of an archaeal aspartate transporter it is now possible to begin to put together a physical picture of how these proteins are able to carry out their dual functions. In this review we shall discuss our current understanding of the functional states of glutamate transporters and how they may arise. We will also discuss some of the alternate conducting states of glutamate transporters and provide definitions of the various states.     

Slips, leaks and channels in glutamate transporters

Glutamate transporters are unusual proteins in that they can function as both a transporter and a chloride channel. With the recent determination of the crystal structure of an archaeal aspartate transporter it is now possible to begin to put together a physical picture of how these proteins are able to carry out their dual functions. In this review we shall discuss our current understanding of the functional states of glutamate transporters and how they may arise. We will also discuss some of the alternate conducting states of glutamate transporters and provide definitions of the various states.     

Bcl-2 family members in the development and degenerative pathologies of the nervous system

Neuronal death is an essential feature in the normal development of the nervous system and in neurodegenerative states of the adult or ageing brain. Bcl-2 is the prototype of a growing family of proteins which control cell death. Many of these proteins are expressed in the nervous system during development and in the adult. Numerous observations have suggested that this family of proteins plays a central role in the control of naturally occurring and pathological neuronal death. In this review, I will discuss the possible mechanisms of action of these proteins as well as their potential use in treating neurodegenerative diseases.     

Conformation-dependent antibodies target diseases of protein misfolding

Many degenerative diseases are fundamentally associated with aging and the accumulation of misfolded proteins as amyloid fibrils. Although such diseases are associated with different proteins, they share several pathological features. These similarities might be due to underlying commonalities in the pathway of aggregation and the structures of the various aggregation products. Because protein misfolding is thought to be central to the pathological state, it is essential to be able to distinguish such pathological states from native and non-pathological states, especially in vivo or in complex mixtures. Conformation-dependent antibodies that specifically recognize misfolded proteins are proving to be useful tools for examining the mechanisms of amyloid formation and for clarifying the roles of various misfolded states in pathogenesis. The common structures and mechanisms hold promise for the development of broad-spectrum drugs and vaccines that will be effective for the treatment of many of these diseases.     

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of DeltaCp of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.     

Role of proteins in protection against radiation-induced damage in membranes

The effect of gamma irradiation on liposomes in the presence of a large number of commercially available proteins has been studied. Experiments were designed to demonstrate that the configuration of both acyl chain and cis C = C bonds created by lipid-protein associations are crucial in autocatalyzed radiation-induced lipid peroxidation. Raman spectroscopy was used to characterize these states. Raman spectra in the C-C stretching region show three prominent bands at 1064, 1090, and 1125 cm-1, assigned to trans, gauche, and trans C-C bonds, respectively. A single symmetrical C = C stretching band assigned to the cis isomer occurs at 1660 cm-1. The intensity ratios (I1064/I1090) and (I1660/I1440) are used as Raman probes to define the conformational states of acyl chains and C = C bonds, respectively. Our data show that the ratio (I1064/I1090) decreases in the presence of proteins, indicating that these proteins induce more gauche structures. Upon irradiation, the ratio (I1064/I1090) increases by about 30% in the absence of proteins and by about 15% in the presence of proteins. This shows that proteins retain the gauche structures in irradiated samples. The ratio (I1660/I1440) decreases in liposomes containing proteins, showing that proteins modify the configuration of cis C = C bonds. Upon irradiation, this ratio decreases by about 45-50% in samples without proteins and by about 10% in samples with proteins. These data show that proteins inhibit the radiation-induced configurational changes in the cis C = C bonds. The determination of radiation-induced peroxides (as malondialdehyde equivalents) in liposomes reveals that proteins inhibit the formation of peroxide products at low molar ratio and that the preventive capacity of different proteins is different. We conclude that proteins alter the conformation of both acyl chains and cis C = C bonds in liposomes and that these altered states are less sensitive to radiation-induced peroxidation.     

Control of cell fate by Hsp70: more than an evanescent meeting

During their lifetime, proteins inevitably expose hydrophobic segments within the polypeptide chains on a molecule's surface, which may be otherwise buried inside the molecules in the proper conformation. This potentially dangerous situation is managed with the aid of the 70-kDa heat shock proteins (Hsp70s) and other molecular chaperones. Although a major function of Hsp70 is assisting in efficient folding of anonymous proteins in unfolded states, recent studies have revealed that Hsp70 plays a variety of specific roles, sometimes deciding the cell fate. These multiple activities are based on the specific binding of Hsp70 to proteins in native states, which regulate cell growth and/or death. It is now well recognized that unfolding of some proteins may cause serious diseases, especially those associated with neurodegeneration, such as Alzheimer's disease. It is suggested that Hsp70 might be a potential drug against these diseases, but caution should be taken because Hsp70 exerts multiple effects by binding to specific proteins.     

Molecular mechanisms of determination in Drosophila.

A number of Drosophila proteins have been identified that play key roles in the establishment of active or inactive states of selector gene expression. Interactions between these proteins and their target selector genes are beginning to be understood, shaping our molecular view as to how stable determination of cells is achieved.     

Improved Gō-like models demonstrate the robustness of protein folding mechanisms towards non-native interactions

The use of simple theoretical models has provided a considerable contribution to our present understanding of the means by which proteins adopt their native fold from the plethora of available unfolded states. A common assumption in building computationally tractable models has been the neglect of stabilizing non-native interactions in the class of models described as "Gō-like." The focus of this study is the characterization of the folding of a number of proteins via a Gō-like model, which aims to map a maximal amount of information reflecting the protein sequence onto a "minimalist" skeleton. This model is shown to contain sufficient information to reproduce the folding transition states of a number of proteins, including topologically analogous proteins that fold via different transition states. Remarkably, these models also demonstrate consistency with the general features of folding transition states thought to be stabilized by non-native interactions. This suggests that native interactions are the primary determinant of most protein folding transition states, and that non-native interactions lead only to local structural perturbations. A prediction is also included for an asymmetrical folding transition state of bacteriophage lambda protein W, which has yet to be subjected to experimental characterization.     

Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques

Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.     

Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins

With the aim of finding non-equilibrium dipole-relaxational electronic excited states of tryptophan residues in proteins the dependence of the fluorescence emission maximum on excitation wavelength was studied for several proteins containing a single tryptophan residue per molecule. Spectral shifts upon red-edge excitation are not observed for short wavelength-emitting proteins (azurin, two-calcium form of whiting parvalbumin, ribonucleases C ₂ and T ₁). This may be because of the non-polar environment of the tryptophan residues in these proteins or because of the absence of dipole-orientational broadening of spectra. The effect was also not found for proteins emitting at long wavelengths (max. at 341–350 nm) —melittin at low ionic strength, IT-Aj1 protease inhibitor, myelin basic protein. In these proteins, the tryptophan residues are exposed to the rapidly relaxing aqueous solvent. Spectral shifts associated with red-edge excitation are observed for proteins emitting in the medium spectral range — human serum albumin in the N and F forms, IT-Aj1 protease inhibitor at pH 2.9, melittin at high ionic strength as well as the albumin-dodecylsulfate complex. This suggests the existence in these proteins of a distribution of microstates for tryptophan environment with various orientation of dipoles and of slow (on the nanosecond time scale) mobility of the field of these dipoles. As a result the emission proceeds from electronic excited states which are not at equilibrium.     

Molecular mechanisms of determination in Drosophila

A number of Drosophila proteins have been identified that play key roles in the establishment of active or inactive states of selector gene expression. Interactions between these proteins and their target selector genes are beginning to be understood, shaping our molecular view as to how stable determination of cells is achieved.     

Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.