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J M Friedman

Publications and source records attributed to J M Friedman.

At least 91 records · Page 5Linked to original sources
PubMed1999

Solution-active structural alterations in liganded hemoglobins C (beta6 Glu --> Lys) and S (beta6 Glu --> Val).

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the alpha and beta chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the alpha1beta2 interface (beta37), and in the A-helix (alpha14/beta15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC approximately HbS > HbA.

Amino Acid Substitution↗
PubMed1999

The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp-->Tyr): origin of the quaternary enhancement effect.

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.

Adult↗
PubMed1999

Epidemiology of neurofibromatosis type 1.

The prevalence of neurofibromatosis type 1 (NF1) is about 1/3,000. There are no known ethnic groups in which NF1 does not occur or is unusually common. The prevalence is somewhat higher in young children than in adults, a difference that probably results at least in part from the early death of some NF1 patients. NF1 is fully penetrant in adults, but many disease features increase in frequency or severity with age. The reproductive fitness of NF1 patients is reduced by about one-half. About half of all cases result from new mutations. The estimated rate of new NF1 mutations is unusually high, but the basis for this high mutation rate is not known. Am. J. Med. Genet. (Semin. Med. Genet.) 89:1-6, 1999.

Adult↗
PubMed1999

Temperature dependent quaternary state relaxation in sol-gel encapsulated hemoglobin.

Samples of human adult hemoglobin (HbA) encapsulated in a wet porous sol-gel are prepared under aerobic and anaerobic conditions. Resonance Raman spectroscopy is used to compare equilibrium deoxyHbA to the nonequilibrium deoxy species generated by deoxygenating an encapsulated oxyHbA sample. The spectra of the deoxygenated samples as a function of delay subsequent to deoxygenation reveal a marked slow down by the gel of the two phases of relaxation: the tertiary relaxation associated with the transition from the liganded R to deoxy R conformations and the quaternary relaxation associated with the deoxy R to deoxy T transition. The temperature dependence (4-80 degrees C) of the relaxation indicates that the internal viscosity of the gel is greatly enhanced at the lower temperatures. At 80 degrees C the tertiary and quaternary relaxations occur over minutes to hours, respectively, whereas at 4 degrees C both relaxations are essentially frozen. These results demonstrate the impressive potential of using sol-gel encapsulation as a means of studying substrate binding induced conformational changes in proteins.

Gels↗
PubMed1999

Interconversion between 3D molecular representations: some macromolecular applications of spherical harmonic-Bessel expansions about an arbitrary center.

An accurate interconversion of three-dimensional molecular spatial information between two alternative orthogonal representations is demonstrated. These alternative descriptions are useful for exhaustive six-dimensional grid searches of empirical energy functions and for estimating the contents of asymmetric units in sparsely packed, non-centrosymmetric crystalline arrays. To illustrate the fidelity of this interconversion, it is implemented to perform an exhaustive grid search in six-dimensional relative rotational and translational space (phase space) with the spherical harmonic-Bessel representation. This search is an extension of Crowther's crystallographic fast rotational overlap function from Patterson space to direct, three-dimensional space. The search uses a three-dimensional spherical harmonic expansion about a single center to represent a probe molecule's shape, charge and van der Waals parameters and numerous complete expansions about single centers to represent the complementary shape, electrostatic potential and van der Waals potential of several target sites about the complementary molecule. The rapid calculation of unique, complete expansions about numerous sites on a grid is made possible by application of the Fourier convolution theorem. Expansions from nearby grid points can be used to reconstruct the same molecular shape accurately, if this shape is contained entirely within the spherical zone of expansion about each grid point. This arbitrariness for the choice of origin, and the accuracy of interconversion between three-dimensional spherical harmonic-Bessel and Fourier representations, suggest a method for describing the contents of non-centrosymmetric, sparsely packed crystals. Incomplete Fourier representations (diffraction amplitudes) provide complex spherical harmonic-Bessel coefficients for a crystal packed with symmetry-enforced, non-centrosymmetrically arranged, non-overlapping spherical expansion zones.

Algorithms↗
PubMed1999

Acute intravenous leptin infusion increases glucose turnover but not skeletal muscle glucose uptake in ob/ob mice.

The mouse ob gene encodes leptin, an adipocyte hormone that regulates body weight and energy expenditure. Leptin has potent metabolic effects on fat and glucose metabolism. A mutation of the ob gene results in mice with severe hereditary obesity and diabetes that can be corrected by treatment with the hormone. In lean mice, leptin acutely increases glucose metabolism in an insulin-independent manner, which could account, at least in part, for some of the antidiabetic effect of the hormone. To investigate further the acute effect of leptin on glucose metabolism in insulin-resistant obese diabetic mice, leptin (40 ng x g(-1) x h(-1)) was administered intravenously for 6 h in C57Bl/6J ob/ob mice. Leptin increased glucose turnover and stimulated glucose uptake in brown adipose tissue (BAT), brain, and heart with no increase in heart rate. A slight increase in all splanchnic tissues was also noticed. Conversely, no increase in skeletal muscle or white adipose tissue (WAT) glucose uptake was observed. Plasma insulin concentration increased moderately but neither glucose, glucagon, thyroid hormones, growth hormone, nor IGF-1 levels were different from phosphate-buffered saline-infused C57Bl/6J ob/ob mice. In addition, leptin stimulated hepatic glucose production, which was associated with increased glucose-6-phosphatase activity. Conversely, PEPCK activity was rather diminished. Interestingly, hepatic insulin receptor substrate (IRS)1-associated phosphatidylinositol 3-kinase activity was slightly elevated, but neither the content of glucose transporter GLUT2 nor the phosphorylation state of the insulin receptor and IRS-1 were changed by acute leptin treatment. Hepatic lipid metabolism was not stimulated during the acute leptin infusion, since the content of triglycerides, glycerol, and citrate was unchanged. These findings suggest that in ob/ob mice, the antidiabetic antiobesity effect of leptin could be the result of a profound alteration of glucose metabolism in liver, BAT, heart, and consequently, glucose turnover. Insulin resistance of skeletal muscle and WAT, while not affected by acute leptin treatment, could also be corrected in the long term and account for some of leptin's antidiabetic effects.

Animals↗
PubMed1998

Leptin and the regulation of body weight in mammals.

The assimilation, storage and use of energy from nutrients constitute a homeostatic system that is essential for life. In vertebrates, the ability to store sufficient quantities of energy-dense triglyceride in adipose tissue allows survival during the frequent periods of food deprivation encountered during evolution. However, the presence of excess adipose tissue can be maladaptive. A complex physiological system has evolved to regulate fuel stores and energy balance at an optimum level. Leptin, a hormone secreted by adipose tissue, and its receptor are integral components of this system. Leptin also signals nutritional status to several other physiological systems and modulates their function. Here we review the role of leptin in the control of body weight and its relevance to the pathogenesis of obesity.

Adipose Tissue↗
PubMed1998

Abnormal regulation of the leptin gene in the pathogenesis of obesity.

A subset of obese humans has relatively low plasma levels of leptin. This finding has suggested that in some cases abnormal regulation of the leptin gene in adipose tissue is etiologic in the pathogenesis of the obese state. The possibility that a relative decrease in leptin production can lead to obesity was tested by mating animals carrying a weakly expressed adipocyte specific aP2-human leptin transgene to C57BL/6J ob/ob mice (which do not express leptin). The transgene does not contain the regulatory elements of the leptin gene and is analogous to a circumstance in which the cis elements and/or trans factors regulating leptin RNA production are abnormal. The ob/ob mice carrying the transgene had a plasma leptin level of 1. 78 ng/ml, which is approximately one-half that found in normal, nontransgenic mice (3.72 ng/ml, P < 0.01). The ob/ob animals expressing the leptin transgene were markedly obese though not as obese as ob/ob mice without the transgene. The infertility as well as several of the endocrine abnormalities generally evident in ob/ob mice were normalized in the ob/ob transgenic mice. However, the ob/ob transgenic mice had an abnormal response when placed at an ambient temperature of 4 degreesC, suggesting that different thresholds exist for the different biologic effects of leptin. Leptin treatment of the ob/ob transgenic mice resulted in marked weight loss with efficacy similar to that seen after treatment of wild-type mice. In aggregate these data suggest that dysregulation of leptin gene can result in obesity with relatively normal levels of leptin and that this form of obesity is responsive to leptin treatment.

Adipocytes↗
PubMed1998

Functional implications of the proximal hydrogen-bonding network in myoglobin: a resonance Raman and kinetic study of Leu89, Ser92, His97, and F-helix swap mutants.

Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme-7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (nu(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to nu(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the nu(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the nu(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the nu(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of nu9 (the 240 cm-1 shoulder on the nu(Fe-His) band) and delta(cbetacccd) at approximately 370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both nu9 and delta(cbetacccd). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in nu(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in gamma7 at approximately 300 cm-1, gamma6 at approximately 335 cm-1, and nu8 at approximately 342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate-rebinding studies on these mutants indicate that disruption of the hydrogen-bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.

Animals↗
PubMed1998

Absence of soluble leptin receptor in plasma from dbPas/dbPas and other db/db mice.

The leptin receptor (Ob-R) is alternatively spliced into at least five different RNAs designated Ob-R(a-e). Ob-R(a-d) predict receptors with a single transmembrane domain, and Ob-Re predicts a secreted form of the receptor. The presence of an approximately 120-kDa soluble leptin receptor in mouse plasma was confirmed by precipitation with leptin-Sepharose beads followed by immunobloting with anti-leptin receptor antibodies. The soluble leptin receptor is larger than that predicted by the primary sequence. Deglycosylation of the receptor with peptide N:glycosidase F results in a decrease in molecular mass to a size consistent with that of the primary sequence. The secreted receptor was present in plasma from wild type mice but was truncated in plasma from db3J/db3J and absent in dbPas/dbPas plasma. Although db3J/db3J mice are known to have a frameshift mutation at amino acid 625, the basis for the mutation in dbPas/dbPas mice was not known. Further studies indicated that dbPas/dbPas mice carry a duplication of exons 4 and 5 of Ob-R. This mutation introduces a premature stop codon into the protein at amino acid 281. The absence of Ob-R in db3J/db3J and dbPas/dbPas mice confirm the identify of the 120-kDa plasma protein as Ob-Re.

Alternative Splicing↗
PubMed1998

A possible allosteric communication pathway identified through a resonance Raman study of four beta37 mutants of human hemoglobin A.

The highly conserved tryptophan at position beta37 occupies a key locus at the hinge region within the alpha1beta2 interface of the mammalian hemoglobins. This residue is thought to play an important role in mediating the heme-heme interaction associated with the cooperative binding of oxygen; however, its explicit function is unclear. In this study, the proximal heme environments of several beta37 mutants of adult human hemoglobin (HbA) are probed using visible (Soret band enhanced) resonance Raman spectroscopy. In the equilibrium deoxy derivatives of these mutants, a systematic variation in proximal strain, as reflected in the iron-proximal histidine (F8) stretching frequency, nu(Fe-His), is seen upon mutation of the beta37 residue. The variation in proximal strain correlates with both the ligand binding rates [Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335] and conformational changes observed at the FG corner through X-ray crystallography [Kavanaugh et al. (1998) Biochemistry 37, 4358-4373]. The results from the deoxy samples indicate a plasticity of the tertiary structure within the T quaternary state. The correlation between the X-ray data and the Raman supports the idea that the proximal strain at the heme within the T state can be modulated by a combination of forces including those arising from the hinge region of the alpha1beta2 interface, from the binding of allosteric effectors, and from the degree of iron displacement from the heme plane. Each of these contributors appears to operate through a shifting of the F helix either away from or toward the FG corner. The Raman spectra obtained from the 10 ns CO photoproduct of the beta37 mutant Hb's indicate that these mutants contain an altered coupling between the R state alpha1beta2 interface and the proximal heme environment. This altered coupling could be due to either dissociation of the ligated mutant tetramers into dimers or the formation of an R state tetramer with significantly weakened hydrogen bonds and van der Waals contacts between the alpha1 and beta2 subunits at the interface. In either case, the results reveal a clear-cut structural basis for the quaternary enhancement effect in which the normal R state quaternary structure produces a higher affinity ligand binding site than that which occurs in the corresponding dimeric form of the protein. The normal R state interface is shown to be important for stabilizing a favorable ligand binding environment that persists long enough after laser photolysis to enhance the geminate rebinding process within the photoproduct. The addition of IHP to the solution of mutant COHb proteins results in photoproduct spectra that are all identical and are consistent with the ligand-bound derivatives having either a T state structure or a very strained and anomalous R state structure.

Allosteric Regulation↗
PubMed1998

Linkage of malignant hyperthermia and hyperkalemic periodic paralysis to the adult skeletal muscle sodium channel (SCN4A) gene in a large pedigree.

Hyperkalemic periodic paralysis (HPP) is caused by mutations of the adult skeletal muscle sodium channel (SCN4A) gene on chromosome 17. Malignant hyperthermia (MH) is a genetically heterogeneous autosomal-dominant disorder occurring in association with various neuromuscular diseases or without other apparent abnormalities. In some families, MH is associated with mutations of a calcium release channel (RYR1) gene on chromosome 19. In other families, linkage of this disorder to the SCN4A gene on chromosome 17 has been suggested. We report on linkage analysis in a family in which both HPP and MH are inherited as autosomal-dominant traits. Two polymorphisms within the SCN4A locus, an RFLP and a (C-A)n repeat, were typed on multiple family members. The findings were consistent with linkage of the polymorphic markers within the SCN4A gene to both HPP (Zmax = 6.79 at theta = 0.0) and MH (Zmax = 1.76 at theta = 0) in this family. Our data provide further evidence that MH is linked to the SCN4A locus in some families.

Adult↗
PubMed1998

Resonance Raman characterization of soluble guanylate cyclase expressed from baculovirus.

Resonance Raman spectra of the alpha 1 beta 1 isoform of bovine lung soluble guanylate cyclase expressed from baculovirus have been measured. The spectra show that the ferric heme is five-coordinate high spin whereas the ferrous heme in the absence of added exogenous ligands is a mixture of six-coordinate low spin and five-coordinate high spin. In the Fe-CO-derivative, the correlation between the Fe-CO frequency (497 cm-1) and the C-O frequency (1959 cm-1) demonstrates that the proximal ligand in our preparation is histidine. The Fe-NO stretching frequency (found at 520 cm-1) and other spectral features of the ferrous Fe-NO-bound sGC are similar to those reported by Deinum et al. (1) and Yu et al. (2). These data indicate that although large preparation-dependent differences in the occupancy of the distal pocket exist, all the preparations have the same proximal histidine ligation and share the same mechanism of activation by NO.

Animals↗
PubMed1998

Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Animals↗