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.     

Nonphotic Phase Shifting in the Old and the Cold

When confined to novel running wheels or when given injections of triazolam in their home cages, old hamsters do not become as active as young hamsters. Therefore, lack of nonphotic phase shifting following such manipulations may stem from insufficient activity or arousal. Phase advances can be obtained in some 10-month-old animals when wheel running during the pulse is increased by the presence of females in estrous condition and in most 18-month-old hamsters by combining confinement to a novel wheel with triazolam injections. These data suggest that there is relatively little if anything wrong in aging hamsters with the nonphotic phase-shifting mechanism itself. The reason why in certain situations old hamsters do not shift appears to be because the nonphotic inputs to these shifting mechanisms are not strong enough. However, when running in novel wheels is increased by carrying out the tests at cold temperatures, most old animals did not show subsequent phase shifts. Evidently it is not running per se that is critical for phase shifts, but probably the motivational context for such running.     

Synthetic heat at mild temperatures

"Synthetic heat", also known as the heat grill illusion, occurs when contact with spatially adjacent warm and cold stimuli produce a sensation of "heat". This phenomenon has been explained as a painful perception that occurs when warm stimulation inhibits cold-sensitive neurons in the spinothalamic tract (STT), which in turn unmasks activity in the pain pathway caused by stimulation of C-polymodal nociceptors (CPNs). The "unmasking model" was tested in experiment 1 by combining warm (35-40°C) and cool ( &#83 27°C) stimuli that were too mild to stimulate CPNs. After discovering that these temperatures produced nonpainful heat, experiment 2 was designed to determine whether heat could be induced when near-threshold cooling was paired with mild warmth, and whether lowering the base temperature for cooling would increase the noxious (burning, stinging) components of heat for fixed cooling steps of 1-3°C. Cooling by just 1°C from a base temperature of 33°C led to reports of heat on more than 1/3 of trials, and cooling by just 3°C evoked heat on 75% of trials. Lowering the base temperature to 31 or 29°C increased reports of heat and burning but did not produce significant reports of pain. Perception of nonpainful heat at such mild temperatures indicates either that cold-sensitive nociceptors with thresholds very similar to cold fibers innervate hairy skin in humans, or that heat can result from integration of warm fiber and cold fiber activity, perhaps via convergence on nonspecific (e.g., WDR) neurons in the STT.     

Growth on Geological Time Scales in the Antarctic Cryptoendolithic Microbial Community

In the process of biogenous weathering of Beacon sandstone in the McMurdo Dry Valleys (Ross Desert), Antarctica, periods of microbial growth, on the time scale of 103-104 years, alternate with sudden exfoliation events. The present study addressed the question of whether microbial growth is continuous between exfoliation events or whether each exfoliation is followed by a period of comparatively rapid growth and then an extended period of steady state. The color intensity (Munsell lightness value) of the rock surface is an indicator of relative age of the crust within the exfoliation cycle, permitting measurement of changes in microbial biomass on a geological time scale. Results indicate that microbial growth is continuous and that exfoliation occurs when the microbial biomass reaches the carrying capacity of the cryptoendolithic habitat.     

Thermal Dissection of Lentil Seedling Amine Oxidase Domains by Differential Scanning Calorimetry

The relationships between the structural and energetic domains of lentil seedling amine oxidase (LSAO) were investigated using modifiers that target the active site and the carbohydrate moiety of the enzyme. An irreversible inhibitor, aminoguanidine, specifically modified the active site of the lentil enzyme, whereas sodium metaperiodate cleaves carbohydrate moieties covalently bound to the native enzyme. Differential scanning calorimetry (DSC) measurements were made on the modified LSAOs. Deconvolution of the reversible thermal DSC profiles of the modified enzyme gave three subpeaks (energetic domains), each of which was assigned to one of the three structural domains of the native protein. Our results led us to conclude that deglycosylation of LSAO has no effect on thermal stability, whereas binding of the inhibitor imparts more stability to the enzyme.     

广西水稻地方品种耐冷性鉴定及相关分析

对419份广西水稻地方品种初级核心种质进行芽期、苗期的耐冷性鉴定及相关分析,结果表明:广西水稻地方品种芽期、苗期耐冷性主要集中在7级和9级,总体耐冷性较弱。芽期、苗期极强耐冷种质(1级)分别为24份和27份,占参试总数的5.73%和6.44%,其中10份种质芽期和苗期均表现极强耐冷(1级)。芽期、苗期耐冷性呈极显著正相关(r=0.66)。粳稻芽期、苗期耐冷性均显著高于籼稻;粘糯稻之间耐冷性差异是由籼粳稻类型的耐冷差异引起的;来自高寒山区稻作区的品种芽期和苗期平均耐冷表现最强。利用34个SSR标记与芽期、苗期耐冷性进行Pearson相关分析,在第7和第9染色体上,各鉴定出1个同时与芽期和苗期耐冷性相关联的位点。本研究为水稻芽期、苗期耐冷育种提供新的抗源材料,并为水稻耐冷基因定位及机理研究奠定基础。     

Plastic responses in the metabolome and functional traits of maize plants to temperature variations

Environmentally inducible phenotypic plasticity is a major player in plant responses to climate change. However, metabolic responses and their role in determining the phenotypic plasticity of plants that are subjected to temperature variations remain poorly understood. The metabolomic profiles and metabolite levels in the leaves of three maize inbred lines grown in different temperature conditions were examined with a nuclear magnetic resonance metabolomic technique. The relationship of functional traits to metabolome profiles and the metabolic mechanism underlying temperature variations were then explored. A comparative analysis showed that during heat and cold stress, maize plants shared common plastic responses in biomass accumulation, carbon, nitrogen, sugars, some amino acids and compatible solutes. We also found that the plastic response of maize plants to heat stress was different from that under cold stress, mainly involving biomass allocation, shikimate and its aromatic amino acid derivatives, and other non‐polar metabolites. The plastic responsiveness of functional traits of maize lines to temperature variations was low, while the metabolic responsiveness in plasticity was high, indicating that functional and metabolic plasticity may play different roles in maize plant adaptation to temperature variations. A linear regression analysis revealed that the maize lines could adapt to growth temperature variations through the interrelation of plastic responses in the metabolomes and functional traits, such as biomass allocation and the status of carbon and nitrogen. We provide valuable insight into the plastic response strategy of maize plants to temperature variations that will permit the optimisation of crop cultivation in an increasingly variable environment.     

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.