Measuring hydrogen exchange rates in invisible protein excited states

Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct ¹⁵N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained.The energy landscape of a protein is a multidimensional surface composed of many local minima in addition to the global minimum that is the native conformation (1, 2). An understanding of the relation between protein structure, dynamics, and function is predicated, therefore, on an analysis of the various conformational states that populate the minima on the landscape. This requires detailed structural and dynamics studies of each of the conformers and quantification of their relative energies as well as the kinetics of exchange between them. Biophysical techniques such as X-ray diffraction and NMR spectroscopy are available for obtaining detailed structural information on the molecules populating the lowest-energy regions of the landscape, providing insight into the structure–function paradigm for a great number of proteins. However, it is becoming increasingly well understood that studies of the ground states of proteins are not sufficient. Additional states that can be sparsely populated and transiently formed, referred to as excited states in what follows, are often important for processes that include molecular recognition, ligand binding, enzyme catalysis, and folding (3–9). Detailed studies of such excited states are, however, challenging because they are not “visible” to standard biophysical methods and as a consequence atomic resolution information is lacking.Recent developments in solution NMR spectroscopy are changing this paradigm by providing an avenue for quantifying excited states at a level of detail that has typically only been possible in studies of highly populated native protein conformers (10). Backbone ¹H, ¹³C, and ¹⁵N chemical shifts for invisible protein states can now be measured so long as they are populated at levels of 0.5% or higher and exchange with an NMR visible state with rates on the order of approximately 100 to several thousand per second (10–12). These chemical shifts have, in turn, been used along with database computational approaches to generate atomic resolution models of excited states (7, 13, 14), providing detailed insights into a range of biochemical processes. It is now possible in some cases to measure scalar and residual dipolar couplings that, in turn, provide additional structural insights (15). Further advances have led to the measurement of side-chain ¹³C and ¹H chemical shifts in invisible states (16–18) that can be sensitive to hydrophobic contacts in these rare conformers. Experiments for quantifying pico- to nanosecond time-scale side-chain dynamics have also emerged (19), showing in some cases large differences in motion between ground and excited states that directly relate to function (20).Although the tool kit of NMR experiments for studying rare protein conformers is expanding, it remains significantly smaller than that for highly populated states, and there is a continuing need for the development of further methodologies. Notably absent from the tool kit is an approach for measuring hydrogen exchange rates in excited-state protein conformers. Ever since the pioneering work of Linderstrøm-Lang in the 1950s it has been recognized that the rates of exchange of amide hydrogens with solvent protons are important site-specific parameters of protein structure and dynamics (21). More recently, amide hydrogen exchange measurements have been used to investigate regions of local protein disorder, folding/unfolding processes, hydrogen bonding, allostery, and ligand binding (22–24). NMR spectroscopy has emerged as a primary tool for quantifying amide hydrogen exchange rates, k_H-EX, on a per-residue basis. However, because the overall transfer of hydrogens from solvent is measured, the extracted rates often contain contributions from all of the exchange-accessible states of the protein (25–27). This is the case for H–D exchange experiments where time-dependent intensity changes of NMR signals are measured to extract k_H-EX (22), as well as experiments that perturb the water signal and quantify the perturbation at exchangeable amide sites (26). Similar bulk measures of k_H-EX are obtained using other methods as well (28), from which insight into a particular excited state can often only be inferred. Herein we describe an approach by which site-specific hydrogen exchange rates of amides in individual states along the energy landscape can be obtained for a protein system at equilibrium and under native conditions. An application to the folding reaction of the G48A Fyn SH3 domain is provided, where k_H-EX values of the native conformation (ground state) and an excited state (unfolded ensemble) are measured. Because exchange rates are quantified by measurement of intensities of cross-peaks in NMR spectra of the ground state, the method is sensitive to k_H-EX values as large as several hundred per second for the excited state. The measurement of “pure” k_H-EX values provides a powerful new approach for understanding the dynamical properties of excited protein states.     

Two mechanisms of ion selectivity in protein binding sites

A theoretical framework is presented to clarify the molecular determinants of ion selectivity in protein binding sites. The relative free energy of a bound ion is expressed in terms of the main coordinating ligands coupled to an effective potential of mean force representing the influence of the rest of the protein. The latter is separated into two main contributions. The first includes all the forces keeping the ion and the coordinating ligands confined to a microscopic subvolume but does not prevent the ligands from adapting to a smaller or larger ion. The second regroups all the remaining forces that control the precise geometry of the coordinating ligands best adapted to a given ion. The theoretical framework makes it possible to delineate two important limiting cases. In the limit where the geometric forces are dominant (rigid binding site), ion selectivity is controlled by the ion-ligand interactions within the matching cavity size according to the familiar "snug-fit" mechanism of host-guest chemistry. In the limit where the geometric forces are negligible, the ion and ligands behave as a "confined microdroplet" that is free to fluctuate and adapt to ions of different sizes. In this case, ion selectivity is set by the interplay between ion-ligand and ligand-ligand interactions and is controlled by the number and the chemical type of ion-coordinating ligands. The framework is illustrated by considering the ion-selective binding sites in the KcsA channel and the LeuT transporter.     

Gut protein uptake and mechanisms of meal-induced cortisol release

The enhanced cortisol release after protein-rich meals might represent a neuroendocrine response to food allergens. We tested whether the antigenicity of proteins contributes to this effect. Twelve healthy men nasogastrically received casein, its less allergenic hydrolysate, and placebo. Contrary to expectations, secretion of cortisol (area under the curve, 742.70 +/- 73.48 vs. 542.95 +/- 70.31 micromol/liter.min, P < 0.03) and ACTH (2020.21 +/- 251.10 vs. 1649.82 +/- 241.23 micromol/liter.min, P < 0.05) was stronger on casein-hydrolysate than casein. Systemic immune activity remained unaffected as indicated by unchanged IL-6 plasma concentrations. This finding indicates that the grade of hydrolysis of a protein and the presence of particular amino acids, rather than its antigenicity, are crucial for the pituitary-adrenal response to nutrients. To further examine whether this response is triggered at the gastrointestinal mucosa or after the substance has reached the circulation, in a supplementary experiment, amino acids were given either nasogastrically or iv to healthy men (n = 4). Only the nasogastric infusion of amino acids induced a significant rise in cortisol concentrations. Serum concentrations of tryptophan, which is known to directly excite the hypothalamo-pituitary-adrenal axis, were comparable for both conditions. We conclude that the meal-related hypothalamo-pituitary-adrenal axis response to amino acids results from a signal that rather acts at the gastrointestinal mucosa than directly via the circulating blood.     

Sodium-calcium ion exchange in cardiac membrane vesicles.

Membrane vesicles isolated from rabbit ventricular tissue rapidly accumulated Ca2+ when an outwardly directed Na+ gradient was formed across the vesicle membrane. Vesicles loaded internally with K+ showed only 10% of the Ca2+ uptake activity observed with Na+-loaded vesicles. Dissipation of the Na+ gradient with the monovalent cation exchange ionophores nigericin or narasin caused a rapid decline in Ca2+ uptake activity. The Ca2+-ionophore A23187 inhibited Ca2+ uptake by Na+-loaded vesicles and enhanced the rate of Ca2+ loss from the vesicles after uptake. Efflux of preaccumulated Ca2+ from the vesicles was stimulated 30-fold by the presence of 50 mM Na+ in the external medium. Na+-dependent uptake and efflux of Ca2+ were both inhibited by La3+. The results indicate that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. Fractionation of the vesicles by density gradient centrifugation revealed a close correspondence between Na+-Ca2+ exchange activity and specific ouabain-binding activity among the various fractions. This relationship suggests that the observed Na+-Ca2+ exchange activity derives from the sarcolemmal membranes within the vesicle preparation.     

Molecular mechanisms of insulin-stimulated glucose uptake in adipocytes

The stimulation of muscle and adipose tissue glucose metabolism, which is ultimately responsible for bringing about post-absorptive blood glucose clearance, is the primary clinically relevant action of insulin. Insulin acts on many steps of glucose metabolism, but one of the most important effects is its ability to increase the rate of cellular glucose transport. This results from the translocation of the insulin-responsive transporter isoform, GLUT4, from intra-cellular vesicular storage sites to the plasma membrane. In adipocytes, a substantial amount of cellular GLUT4 is located in a specific highly insulin-responsive storage pool, termed GLUT4 Storage Vesicles (GSVs). GLUT4 can also translocate to the plasma membrane from the recycling endosomal pool which also additionally contains the GLUT1 isoform of glucose transporter and the transferrin receptor. In this article we review the molecular mechanism by which insulin stimulates GLUT4 translocation in adipose cells, including the nature of the signaling pathways involved and the role of the cytoskeleton.     

Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia

Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.     

Fasting decreases rates of noninsulin-mediated glucose uptake in man

Although fasting decreases insulin-mediated glucose uptake (IMGU), its effect on noninsulin-mediated glucose uptake (NIMGU) is not known. To examine this issue we studied seven obese men [mean (+/- SD) age, 36 +/- 5 yr; weight, 91 +/- 13 kg] after an overnight fast (day 0) and 3 days (day 4) and 9 days (day 10) of total fasting and six normal weight men (age, 32 +/- 4 yr; weight, 73 +/- 6 kg) after an overnight and 3 days of fasting. To study NIMGU, somatostatin (0.16 micrograms/kg.min) was infused to create severe insulin deficiency and [3H]3-glucose to measure glucose disappearance (Rd), while serum glucose was sequentially clamped at a level of about 4.7 mmol/L for 180 min and about 11 mmol/L for an additional 100 min. The results from the last 60 min of each glycemic plateau were used for analysis. Under these conditions insulin action is absent and Rd = NIMGU. Since under conditions of euglycemic insulinopenia, NIMGU into noncentral nervous system tissues is negligible, and central nervous system (CNS) glucose uptake saturates at physiological glucose concentrations, it follows that at a glucose level of about 4.7 mmol/L, NIMGU reflects CNS glucose uptake and at about 11 mmol/L, NIMGU reflect CNS plus non-CNS tissues. Thus, non-CNS NIMGU = NIMGU at 11 mmol/L - NIMGU at about 4.7 mmol/L. The obese subjects' mean weight fell to 88 +/- 5 kg on day 4 and 85 +/- 5 kg on day 10 (P less than 0.001 between all values). The mean basal serum glucose level fell from 5.3 +/- 0.1 on day 0 to 4.2 +/- 0.2 and 3.8 +/- 0.2 mmol/L on days 4 and 10, respectively (P less than 0.01 between all values). During insulinopenia plasma FFA and serum beta-hydroxybutyrate levels on day 10 were 3- and 30-fold higher than the basal prefast levels, respectively. Noninsulin-mediated glucose clearance at about 4.7 mmol/L did not change during fasting [0.0016 +/- 0.0001 (day 0) vs. 0.0016 +/- 0.0001 (day 4) and 0.0014 +/- 0.0001 L/kg.min (day 10); P = NS between all values]; at about 11 mmol/L noninsulin-mediated glucose clearance fell from 0.0016 +/- 0.0001 on day 0 to 0.0001 +/- 0.0001 dL/kg.min on day 10 (P less than 0.001). Results in the lean group were similar to those in the obese group after a 3-day fast.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glucocorticoids enhance somatomedin-C binding and stimulation of amino acid uptake in human fibroblasts

The regulation of growth by the GH-dependent mitogen somatomedin-C (Sm-C) may involve not only changes in circulating levels of Sm-C, but also alterations in cellular sensitivity to Sm-C induced by humoral factors. Glucocorticoids have been reported to enhance the stimulatory effect of Sm-C on DNA synthesis and cell replication in cultured human fibroblasts, but the cellular alteration responsible for this effect was not fully defined. Using an assay for cellular sensitivity to Sm-C based on stimulation of uptake of the amino acid analog aminoisobutyric acid, a 20-h preincubation with 100 nM dexamethasone was found to enhance both the sensitivity and the maximal responsiveness of human fibroblasts to a 3-h incubation with Sm-C. This effect was found using fibroblasts from multiple normal donors of different ages, and dexamethasone was approximately 10-fold more potent than hydrocortisone. The glucocorticoid antagonist RU486 (100 nM) largely reversed the enhancing effect of 100 nM dexamethasone on Sm-C action. Binding of [125I]Sm-C to intact fibroblast monolayers or trypsin-dispersed cells could be increased by 60-80% by glucocorticoid preincubation, and this increase correlated well with enhanced stimulation of [3H]aminoisobutyric acid uptake, suggesting that the enhancement of Sm-C action in glucocorticoid-treated cells may be mediated at the level of the Sm-C receptor.     

Exploring knotting mechanisms in protein folding

One of the most striking topological features to be found in a protein is that of a distinct knot formed by the path of the polypeptide backbone. Such knotted structures represent some of the smallest “self-tying” knots observed in Nature. Proteins containing a knot deep within their structure add an extra complication to the already challenging protein-folding problem; it is not obvious how, during the process of folding, a substantial length of polypeptide chain manages to spontaneously thread itself through a loop. Here, we probe the folding mechanism of YibK, a homodimeric α/β-knot protein containing a deep trefoil knot at its carboxy terminus. By analyzing the effect of mutations made in the knotted region of the protein we show that the native structure in this area remains undeveloped until very late in the folding reaction. Single-site destabilizing mutations made in the knot structure significantly affect only the folding kinetics of a late-forming intermediate and the slow dimerization step. Furthermore, we find evidence to suggest that the heterogeneity observed in the denatured state is not caused by isomerization of the single cis proline bond as previously thought, but instead could be a result of the knotting mechanism. These results allow us to propose a folding model for YibK where the threading of the polypeptide chain and the formation of native structure in the knotted region of the protein occur independently as successive events.     

The mechanisms of iron uptake by fetal rat hepatocytes in culture

The mechanisms of iron accumulation by cultured hepatocytes isolated from fetal rat liver (19 days gestation) were investigated using rat transferrin labeled with 125I and 59Fe. The rates of iron and transferrin internalization by the cells were measured by incubating the hepatocytes with the labeled transferrin at 37 degrees C followed by treatment with pronase at 4 degrees C to remove surface-bound transferrin and iron. Iron internalization increased linearly with time. Approximately 65% of the internalized iron was incorporated into ferritin. In contrast to iron, the rate of transferrin internalization was biphasic, with a rapid phase during the first 10 to 15 min and a second slower phase which becomes more apparent after that time. Iron and transferrin internalization were temperature-dependent. Chase experiments showed that the internalized transferrin donated all of its iron to the cell and was then released in a biphasic manner which was dependent on the time of preincubation with radiolabeled transferrin. These experiments showed that iron uptake occurs by at least three processes. The first mechanism involves the specific receptor-mediated endocytosis of transferrin. Each cell has an average of 7.8 +/- 1.0 X 10(5) (mean +/- SE, n = 5) transferrin binding sites with an apparent association constant of 2.0 +/- 0.4 X 10(6) M-1. The second process is nonsaturable up to a transferrin concentration of at least 6 microM but like the specific process, also leads to accumulation of iron in excess of transferrin. It involves the endocytosis of transferrin mediated by 4.2 X 2.6 X 10(5) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two size-selective mechanisms specifically trap bacteria-sized food particles in Caenorhabditis elegans

Caenorhabditis elegans is a filter feeder: it draws bacteria suspended in liquid into its pharynx, traps the bacteria, and ejects the liquid. How pharyngeal pumping simultaneously transports and filters food particles has been poorly understood. Here, we use high-speed video microscopy to define the detailed workings of pharyngeal mechanics. The buccal cavity and metastomal flaps regulate the flow of dense bacterial suspensions and exclude excessively large particles from entering the pharynx. A complex sequence of contractions and relaxations transports food particles in two successive trap stages before passage into the terminal bulb and intestine. Filtering occurs at each trap as bacteria are concentrated in the central lumen while fluids are expelled radially through three apical channels. Experiments with microspheres show that the C. elegans pharynx, in combination with the buccal cavity, is tuned to specifically catch and transport particles of a size range corresponding to most soil bacteria.     

Lipid-protein interaction in the phosphatidylcholine exchange protein.

Incorporation of 2-acyl spin-labeled lecithin into the phosphatidylcholine protein from bovine liver results in an immobilization of the spin-label at the methyl and the carboxyl terminal end of the acyl chain. The nitroxide group on the protein-bound lecithin molecule is not accessible to ascorbate. This suggests that lecithin is buried in a pocket on the protein, which effectively shields the acyl chains from the medium.     

Isoproterenol does not enhance Ca-dependent Na/Ca exchange current in intact rabbit ventricular myocytes

Cardiac Na/Ca exchange (NCX, NCX1.1) is critical in cardiac myocyte Ca regulation, and its altered function contributes to inotropic state, systolic dysfunction in heart failure and arrhythmogenesis. Regulation of NCX is multifaceted, but protein kinase A (PKA) effects on NCX function are controversial. Here, we use three different and complementary approaches to compare NCX function +/-1 microM isoproterenol (ISO) in intact rabbit cardiac myocytes (in paired comparisons). First, in field-stimulated intact cells we inferred the cytosolic [Ca] ([Ca](i)) dependence of NCX function from the decay rate of caffeine-induced [Ca](i) transients. Second, we measured caffeine-induced [Ca](i) and inward I(NCX) simultaneously (perforated patch voltage clamp), to measure directly the [Ca](i) dependence of NCX rate. Third, using whole cell ruptured patch with [Ca](i) heavily buffered to 100 nM, [Na](i)=10 mM, and I(Ca), SR Ca release and Na/K pump all blocked, we recorded I(NCX) ramps at 37 degrees C. We find that NCX function is not altered by PKA activation under any of these three protocols, where intracellular conditions ranged from near-physiological to highly controlled. This does not rule out NCX modulation by PKA under all conditions, or in species other than rabbit. However, such effects are likely to be either minor (vs. other PKA actions on myocyte Ca handling) or indirect, such as secondary effects dependent on altered local [Ca](i) and [Na](i).     

Social capital strategies to enhance hepatitis C treatment awareness and uptake among men in prison

Prisoner populations are characterized by high rates of hepatitis C (HCV), up to thirty times that of the general population in Australia. Within Australian prisons, less than 1% of eligible inmates access treatment. Public health strategies informed by social capital could be important in addressing this inequality in access to HCV treatment. Twenty‐eight male inmates participated in qualitative interviews across three correctional centres in New South Wales, Australia. All participants had recently tested as HCV RNA positive or were receiving HCV treatment. Analysis was conducted with participants including men with experiences of HCV treatment (n=10) (including those currently accessing treatment and those with a history of treatment) and those who were treatment naïve (n=18). Social capital was a resourceful commodity for inmates considering and undergoing treatment while in custody. Inmates were a valuable resource for information regarding HCV treatment, including personal accounts and reassurance (bonding social capital), while nurses a resource for the provision of information and care (linking social capital). Although linking social capital between inmates and nurses appeared influential in HCV treatment access, there remained opportunities for increasing linking social capital within the prison setting (such as nurse‐led engagement within the prisons). Bonding and linking social capital can be valuable resources in promoting HCV treatment awareness, uptake and adherence. Peer‐based programmes are likely to be influential in promoting HCV outcomes in the prison setting. Engagement in prisons, outside of the clinics, would enhance opportunities for linking social capital to influence HCV treatment outcomes.     

Molecular imaging in neuroendocrine tumors: Molecular uptake mechanisms and clinical results

Neuroendocrine tumors can originate almost everywhere in the body and consist of a great variety of subtypes. This paper focuses on molecular imaging methods using nuclear medicine techniques in neuroendocrine tumors, coupling molecular uptake mechanisms of radiotracers with clinical results. A non-systematic review is presented on receptor based and metabolic imaging methods. Receptor-based imaging covers the molecular backgrounds of somatostatin, vaso-intestinal peptide (VIP), bombesin and cholecystokinin (CCK) receptors and their link with nuclear imaging. Imaging methods based on specific metabolic properties include meta-iodo-benzylguanide (MIBG) and dimercapto-sulphuric acid (DMSA-V) scintigraphy as well as more modern positron emission tomography (PET)-based methods using radio-labeled analogues of amino acids, glucose, dihydroxyphenylalanine (DOPA), dopamine and tryptophan. Diagnostic sensitivities are presented for each imaging method and for each neuroendocrine tumor subtype. Finally, a Forest plot analysis of diagnostic performance is presented for each tumor type in order to provide a comprehensive overview for clinical use.