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L E Kay

Publications and source records attributed to L E Kay.

At least 91 records · Page 5Linked to original sources
PubMed1995

Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein.

Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase. Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.

Amino Acid Sequence↗
PubMed1995

Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer.

Two-dimensional NMR 15N relaxation studies have been used to characterize the backbone dynamics and folding transition of the N-terminal SH3 domain of the protein drk (drkN SH3). The isolated drkN SH3 domain exists in equilibrium between a folded and an unfolded state in aqueous buffer and near neutral pH [Zhang et al. (1994) J. Biomol. NMR 4, 845]. The backbone dynamics of both the folded and unfolded states in this exchanging system have been determined and the rates of the folding transition estimated at 14 degrees C. For 12 residues, the values of the spectral density functions of the backbone amide bond vectors at a number of frequencies have been established. Results show that while the unfolded state has considerably greater dynamic behavior, the overall motional properties are consistent with it having a reasonably compact structure in solution. In contrast to the folded state, considerable variations are seen in the values of the spectral densities of the unfolded state as a function of residue number; these variations do not appear to be strongly correlated with structural elements in the folded state. The mean value of the exchange rate associated with the folding transition was determined to be 0.89 s-1, similar to previous measurements of the rate of formation of beta-structure.

Amino Acid Sequence↗
PubMed1995

Spectral density function mapping using 15N relaxation data exclusively.

A method is presented for the determination of values of the spectral density function, J(omega), describing the dynamics of amide bond vectors from 15N relaxation parameters alone. Assuming that the spectral density is given by the sum of Lorentzian functions, the approach allows values of J(omega) to be obtained at omega = 0, omega N and 0.870 omega H, where omega N and omega H are Larmor frequencies of nitrogen and proton nuclei, respectively, from measurements of 15N T1, T2 and 1H-15N steady-state NOE values at a single spectrometer frequency. Alternatively, when measurements are performed at two different spectrometer frequencies of i and j MHz, J(omega) can be mapped at omega = 0, omega iN, omega jN, 0.870 omega iH and 0.870 omega iH, where omega iN, for example, is the 15N Larmor frequency for a spectrometer operating at 1 MHz. Additionally, measurements made at two different spectrometer frequencies enable contributions to transverse relaxation from motions on millisecond-microsecond time scales to be evaluated and permit assessment of whether a description of the internal dynamics is consistent with a correlation function consisting of a sum of exponentials. No assumptions about the specific form of the spectral density function describing the dynamics of the 15N-NH bond vector are necessary, provided that dJ(omega)/d omega is relatively constant between omega = omega H + omega N to omega = omega H - omega N. Simulations demonstrate that the method is accurate for a wide range of protein motions and correlation times, and experimental data establish the validity of the methodology. Results are presented for a folded and an unfolded form of the N-terminal SH3 domain of the protein drk.

Animals↗
PubMed1995

Field gradient techniques in NMR spectroscopy.

In recent years NMR spectroscopy has emerged as a powerful technique for studying the structure and dynamics of biomolecules in solution. The development of shielded pulsed-field gradient coils for high-resolution NMR spectroscopy has led to significant improvements in a large number of experiments which are used to provide such information. Experiments enhanced with pulsed-field gradients have fewer artifacts, suffer far less from problems of solvent suppression and have reduced phase cycles relative to their non-gradient counterparts. The whole array of NMR experiments available for macromolecular structure determination is likely to benefit substantially from the incorporation of gradient pulses.

Diffusion↗
PubMed1995

Who wrote the book of life? Information and the transformation of molecular biology, 1945-55.

This paper focuses on the opening of a discursive space: the emergence of informational and scriptural representations of life and hereditiy and their self-negating consequences for the construction of biological meaning. It probes the notion of writing and the book of life and shows how molecular biology's claims to a status of language and texuality undermines its own objective of control. These textual significations were historically contingent. The informational representations of heredity and life were not an outcome of the internal cognitive momentum of molecular biology; they were not a logical necessity of the unravelling of the base-pairing of the DNA double-helix. They were transported into molecular biology still within the protein paradigm of the gene in the 1940s and permeated nearly every discipline in the life and social sciences. These information-based models, metaphors, linguistic, and semiotic tools which were central to the formulation of the genetic code were transported into molecular biology from cybernetics, information theory, electronic computing, and control and communication systems--technosciences that were deeply embedded with the military experiences of World War II and the Cold War. The information discourse thus became fixed in molecular biology not because it worked in the narrow epistemic sense (it did not), but because it positioned molecular biology within postwar discourse and culture, perhaps within the transition to a post-modern information-based society.

History, 20th Century↗
PubMed1995

Adolescent sexual intercourse. Strategies for promoting abstinence in teens.

As sex, drugs, and rock 'n' roll have given way to STDs, HIV, and AIDS, adolescent sex has become an increasingly high-risk behavior. Dr Kay strongly believes that primary care physicians can have a prominent role in educating teens and their parents about the consequences of premature sexual activity and in promoting healthy living. In this article, he presents the case against adolescent sexual intercourse and outlines a concrete approach to dealing with adolescent patients at risk and their families.

Adolescent↗
PubMed1994

A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular complexes.

A pulsed field gradient three-dimensional isotope-filtered 13C HMQC-NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp-repressor, its natural abundance co-repressor, L-tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra- and intermolecular NOEs in the isotope-edited 3D 13C HMQC-NOESY spectrum of the complex.

Bacterial Proteins↗
PubMed1994

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Amino Acid Sequence↗
PubMed1994

Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed with a high affinity binding peptide.

The solution structure of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLC-gamma 1), in complex with a phosphopeptide corresponding to its Tyr-1021 high affinity binding site on the platelet-derived growth factor receptor, has been determined by nuclear magnetic resonance spectroscopy. The topology of the SH2-phosphopeptide complex is similar to previously reported Src and Lck SH2 complexes. However, the binding site for residues C-terminal to the phosphotyrosine (pTyr) is an extended groove that contacts peptide residues at the +1 to +6 positions relative to the pTyr. This striking difference from Src and Lck reflects the fact that the PLC-gamma 1 complex involves binding of a phosphopeptide with predominantly hydrophobic residues C-terminal to the pTyr and therefore serves as a prototype for a second class of SH2-phosphopeptide interactions.

Amino Acid Sequence↗
PubMed1994

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy.

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.

Amino Acid Sequence↗
PubMed1994

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques.

The backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.

Amino Acid Sequence↗
PubMed1994

A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium.

A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, an approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.

Chemical Phenomena↗
PubMed1992

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Leucine↗
PubMed1992

Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible.

The backbone dynamics of Ca(2+)-saturated recombinant Drosophila calmodulin has been studied by 15N longitudinal and transverse relaxation experiments, combined with 15N(1H) NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.5-0.6 range. The anisotropy observed in the motion of the two globular calmodulin domains is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that, for the purposes of 15N relaxation, the tumbling of the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of calmodulin is effectively independent. A slightly shorter motional correlation time (tau c approximately 6.3 ns) is obtained for the C-terminal domain compared to the N-terminal domain (tau c approximately 7.1 ns), in agreement with the smaller size of the C-terminal domain. A high degree of mobility, with order parameters of approximately 0.5, is also observed in the loop that connects the first with the second EF-hand type calcium binding domain and in the loop connecting the third and fourth calcium binding domain.

Animals↗
PubMed1991

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy.

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.

Alanine↗