Quantifying energetic contributions to ground state destabilization

Solution differential scanning calorimetry (DSC) of oxidized amicyanin, a Type I copper protein, at pH 7.5 reveals two thermal transitions. The major transition at 67.7 degrees C corresponds to the disruption of the Cys(92) thiolate to Cu(II) charge transfer as evidenced by a corresponding temperature-dependent loss of amicyanin visible absorbance. A minor transition at 75.5 degrees C describes the further irreversible protein unfolding. Reduced amicyanin exhibits a pH-dependent change of the copper ligand geometry. At pH 8.5 where the Type I tetrahedral geometry is maintained, DSC reveals two thermal transitions with T(m) values similar to that of oxidized amicyanin. At pH 6.2 where the Cu(I) coordination is trigonal planar, reduced amicyanin exhibits a single thermal transition with a lower T(m) of 64.0 degrees C. Apoamicyanin, from which copper has been removed, also exhibits a single thermal transition but with a much lower T(m) of 51.8 degrees C. Thus, the thermal stability of amicyanin is dictated both by the presence or absence of copper and its ligand geometry, but not its redox state. The physiological relevance of these data is discussed.     

Molecular dynamics simulation of the C-terminal sterile alpha-motif domain of human p73alpha: evidence of a dynamical relationship between helices 3 and 5

We used molecular dynamics simulation to evaluate the association properties of C-terminal sterile alpha-motif (SAM) domain of human p73alpha. To test the dimerization propensity of this structure we carried out four simulations: EphB2 X-ray dimer, p73 modeled dimer, p73 NMR monomer, and p73 modeled monomer with an elongated helix 5. The results show a direct interaction between helix 5 and helix 3 since a conformational collapse of helix 3 is observed when dimer contact and/or an elongation of helix 5 is introduced by modeling in p73 SAM domain. On the basis of these results we suggest that the recognition properties of the SAM domains may be modulated by the conformational state of helix 5.     

Non-Aggregating Amylin Fragments as an Inhibitors of the Aggregation Process of Susceptible to Aggregation Fragments 18–22, 23–27, and 33–37 of Hormone

Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1–7, 8–12, 13–17 and 28–32 of amylin would inhibit the aggregation of the amyloidogenic cores 18–22, 23–27, 33–37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1–7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18–22, 23–27, 33–37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1–7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.     

Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm

The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (−BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., −BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.     

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile α-motif (SAM) domain (residues 505–579), a region crucial to explaining the human genetic disease ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of free-dom of individual residues and discussion of the possible causes for the pathology.     

A Raman-active competitive inhibitor of OMP decarboxylase

6-Cyanouridine 5'-phosphate was shown to act as a competitive inhibitor of yeast OMP decarboxylase, with a K(i) value of 1.1 x 10(-5)M. Upon binding by the active site of yeast OMP decarboxylase (EC 4.1.1.23), the Raman stretching frequency of the nitrile group of 6-cyanouridine 5'-phosphate decreases from 2240 to 2225 cm(-1). Based on the behavior of a model compound, 6-cyano-1,3-dimethyluracil, and on vibrational calculations, the observed change in stretching frequency is attributed to desolvation of the ligand, and distortion of the ligand in which the nitrile group moves out of the plane of the pyrimidine ring. Similar distortions may play a role in substrate activation by OMP decarboxylase, contributing to the catalytic process.     

Adenosine triphosphate energy‐independently controls protein homeostasis with unique structure and diverse mechanisms

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid–liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2–12 mM, much higher than required for its previously‐known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic‐acid‐binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding‐induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π‐π, π‐cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy‐independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein‐aggregation diseases.     

Diverse role of conformational dynamics in carboxypeptidase A-driven peptide and ester hydrolyses: disclosing the "perfect induced fit" and "protein local unfolding" pathways by altering protein stability

Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of "initial rates" is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus "active" product) concentration, made it possible to rigorously determine the physically meaningful "effective" values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S(3) subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the "promoted water" mechanism for the peptide substrate that, seemingly, provides the almost "perfect induced fit" (low-barrier conformational adaptation), and (b) presumably, the "anhydride intermediate" mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, "partial or local unfolding") of the protein environment in the course of the rate-determining step.     

The epidemiological consequences of immune priming

Exposure to low doses of pathogens that do not result in the host becoming infectious may ‘prime’ the immune response and increase protection to subsequent challenge. There is increasing evidence that such immune priming is a widespread and important feature of invertebrate host–pathogen interactions. Immune priming clearly has implications for individual hosts but will also have population-level implications. We present a susceptible–primed–infectious model—in contrast to the classic susceptible–infectious–recovered framework—to investigate the impacts of immune priming on pathogen persistence and population stability. We describe impacts of immune priming on the epidemiology of the disease in both constant and seasonal environments. A key result is that immune priming may act to destabilize population dynamics. In particular, when the proportion of individuals becoming primed rather than infected is high, but this priming does not confer full immunity, the population may be strongly destabilized through the generation of limit cycles. We discuss the implications of our model both in the context of invertebrate immunity and more widely.