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K J Lumb

Publications and source records attributed to K J Lumb.

13 recordsLinked to original sources
PubMed2000

Reevaluation of transcriptional regulation by TATA-binding protein oligomerization: predominance of monomers.

The TATA-binding protein (TBP) plays an important role in transcriptional initiation by all three nuclear RNA polymerases. TBP contains a conserved C-terminal domain (cTBP) that binds DNA. Crystallographic studies of cTBP (i.e., TBP without the N-terminal domain) from various species and molecular biology studies of cTBP and mixed cTBP/TBP species have led to the view that DNA binding by TBP is regulated by TBP dimerization. Using sedimentation equilibrium, we show that yeast cTBP forms dimers in solution at 5 degrees C with a dissociation constant of 7 +/- 1 microM. This observation of cTBP dimers in solution is in accord with the dimeric state observed in crystal structures of cTBP. In contrast, physiologically relevant, full-length yeast TBP is monomeric at 5 degrees C and forms dimers at 30 degrees C with a dissociation constant of 51 +/- 16 microM. This dissociation constant precludes formation of stable full-length TBP dimers at physiological concentrations. In addition, we tested for yeast TBP oligomerization in the presence of TBP-associated factors in the context of TFIID. No evidence for TBP oligomers was found using immunoprecipitation techniques from yeast whole-cell extracts. We conclude that yeast TBP is predominantly monomeric under physiological conditions, arguing against a role for TBP dimerization in the regulation of transcriptional initiation.

DNA-Binding Proteins↗
PubMed2000

Intrinsic structural disorder of the C-terminal activation domain from the bZIP transcription factor Fos.

The bZIP proto-oncoprotein c-Fos activates transcription of a wide variety of genes involved in cell growth. The C-terminal activation domain of c-Fos is functionally independent of the remainder of the protein. Fos-AD corresponds to the C-terminal activation domain of human c-Fos (residues 216-380). Fos-AD suppresses (squelches) transcription in vitro, as expected for a functional activation domain lacking a DNA-binding domain. Fos-AD is unstructured and highly mobile, as demonstrated by circular dichroism spectra indicative of unfolded proteins, a lack of (1)H chemical shift dispersion, and negative (1)H-(15)N heteronuclear nuclear Overhauser effects. The hydrodynamic properties of Fos-AD are also consistent with an extended structure. We conclude that the C-terminal domain of human c-Fos is biologically active yet intrinsically disordered. Our results suggest that conformational disorder is an integral aspect of the diverse contributions to transcriptional regulation by c-Fos.

Basic-Leucine Zipper Transcription Factors↗
PubMed1999

Random-coil chemical shifts of phosphorylated amino acids.

The 1H, 13C, 15N and 31P random-coil chemical shifts and phosphate pKa values of phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH2 have been obtained in water at 25 degrees C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.

Amino Acids↗
PubMed1995

A buried polar interaction imparts structural uniqueness in a designed heterodimeric coiled coil.

Buried polar residues are a common feature of natural proteins. ACID-p1 and BASE-p1 are two designed peptides that form a parallel, heterodimeric coiled coil with a fixed tertiary structure [O'Shea, E. K., Lumb, K. J., & Kim, P. S. (1993) Curr. Biol. 3, 658-667]. The interface between the ACID-p1 and BASE-p1 helices consists of hydrophobic Leu residues, with the exception of a single polar residue, Asn 14. In the crystal structure of the GCN4 leucine zipper coiled coil, an analogous Asn is hydrogen bonded to the corresponding Asn of the opposing helix, thereby forming a buried polar interaction in an otherwise hydrophobic interface between the helices [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. This buried polar interaction in the ACID-p1/BASE-p1 heterodimer was removed by substituting Asn 14 with Leu. The Asn 14-->Leu variants are significantly more stable than the p1 peptides and preferentially form a heterotetramer instead of a heterodimer. Strikingly, the heterotetramer does not fold into a unique structure; in particular, the helices lack a unique orientation. Thus, the Asn 14 residue imparts specificity for formation of a two-stranded, parallel coiled coil at the expense of stability. The results suggest that, whereas nonspecific hydrophobic interactions contribute to protein stability, the requirement to satisfy the hydrogen bonding potential of buried polar residues in the generally hydrophobic environment of the protein interior can impart specificity (structural uniqueness) to protein folding and design.

Amino Acid Sequence↗
PubMed1995

Measurement of interhelical electrostatic interactions in the GCN4 leucine zipper.

The dimerization specificity of the bZIP transcription factors resides in the leucine zipper region. It is commonly assumed that electrostatic interactions between oppositely charged amino acid residues on different helices of the leucine zipper contribute favorably to dimerization specificity. Crystal structures of the GCN4 leucine zipper contain interhelical salt bridges between Glu20 and Lys15' and between Glu22 and Lys27'. 13C-nuclear magnetic resonance measurements of the glutamic acid pKa values at physiological ionic strength indicate that the salt bridge involving Glu22 does not contribute to stability and that the salt bridge involving Glu20 is unfavorable, relative to the corresponding situation with a neutral (protonated) Glu residue. Moreover, the substitution of Glu20 by glutamine is stabilizing. Thus, salt bridges will not necessarily contribute favorably to bZIP dimerization specificity and may indeed be unfavorable, relative to alternative neutral-charge interactions.

Amino Acid Sequence↗
PubMed1995

Cooperatively folded proteins in random sequence libraries.

The structural properties of proteins recovered from random sequence libraries can be used to investigate the relationship between folding and sequence information. Here, we show that helical proteins displaying cooperative thermal denaturation transitions can be easily recovered from a library containing 80-residue proteins predominantly composed of glutamine, leucine, and arginine, with an average hydrophobicity level similar to that of natural proteins. The native structure of one of these proteins has a stability and oligomeric form similar to that of many natural proteins but differs in having no slowly exchanging amide hydrogens.

Amino Acid Sequence↗
PubMed1994

Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4.

One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation. However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation. A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A. (1991) Science 254, 539-544]. Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form. N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7). In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length. Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities. These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.

Amino Acid Sequence↗
PubMed1994

Formation of a hydrophobic cluster in denatured bovine pancreatic trypsin inhibitor.

Bovine pancreatic trypsin inhibitor (BPTI) unfolds upon reduction of its three disulfide bonds. A recombinant model of the reduced state of BPTI, called [R]Ala, in which all six Cys residues are replaced with Ala, has been expressed in Escherichia coli. 1H nuclear magnetic resonance spectroscopy shows that [R]Ala does not contain stable secondary structure. Some chemical shift dispersion exists, however, suggesting the existence of non-random interactions in [R]Ala. In particular, the side-chain protons of Ile19 resonate upfield of those of Ile18. This observation was investigated using an eight residue peptide model, P17-24, corresponding to residues 17 to 24 of BPTI. The non-random chemical shift dispersion of the Ile residues observed in [R]Ala also occurs in P17-24, indicating that P17-24 contains interactions that are similar to those found in the corresponding region of [R]Ala. The only interresidue nuclear Overhauser effects observed in P17-24 are between the ring protons of Tyr21 and the gamma CH3 group of Ile19, indicating that these protons are in close proximity. Substitution of Tyr21 by Ala in P17-24 results in the loss of the chemical shift dispersion of the Ile resonances, suggesting that the upfield shifts of the Ile19 resonances are due to ring current shifts arising from the proximity of Tyr21. Collectively, these results suggest that the side-chain of Ile19 is positioned at least some of the time above the plane of the aromatic ring of Tyr21. We conclude that these two residues participate in a hydrophobic cluster in P17-24 and in the denatured state of BPTI.

Alanine↗
PubMed1994

1H nuclear magnetic resonance studies of hen lysozyme-N-acetylglucosamine oligosaccharide complexes in solution. Application of chemical shifts for the comparison of conformational changes in solution and in the crystal.

Two-dimensional 1H nuclear magnetic resonance spectroscopy has been used to examine the complexes formed in solution between hen egg-white lysozyme and N-acetylglucosamine (GlcNAc) oligosaccharides. Changes in chemical shift have been measured for resonances of the majority of residues of lysozyme on binding the monomer, dimer and trimer of GlcNAc. The three inhibitors induce very similar changes in chemical shift, and these increase slightly with the length of the oligosaccharide. The largest changes are confined principally to the vicinity of site C in the active site cleft of the enzyme. These changes in chemical shift have been compared with differences in the ring current chemical shifts calculated from the crystal structures of unbound and GlcNAc3 bound lysozyme. This comparison suggests that the major conformational changes of residues in the vicinity of site C of the enzyme, that are caused by the binding of GlcNAc3, observed in the diffraction studies are at least consistent with the changes that occur in solution. Small changes in chemical shift are observed in the enzyme in regions remote from the active site, which indicate that the effects of inhibitor binding are felt throughout the enzyme. These changes in chemical shift correlate to a lesser extent than those near site C with the changes in chemical shift predicted from changes in conformation observed in the crystal structures. The results illustrate that chemical shifts are useful in assessing the significance of small conformational changes in proteins, although the usefulness of this approach will be limited by the resolution of the crystallographic structures, as well as the uncertainties in the origins of the chemical shift. Although conformational changes in site C account for many of the changes in the NMR spectrum of lysozyme, evidence is, however, presented for multiple binding sites for the GlcNAc oligosaccharides in solution, perhaps involving partial occupancy of site D.

Acetylglucosamine↗
PubMed1992

1H nuclear magnetic resonance studies of the interaction of urea with hen lysozyme. Origins of the conformational change induced in hen lysozyme by N-acetylglucosamine oligosaccharides.

The interaction between hen lysozyme and urea has been investigated using 1H nuclear magnetic resonance spectroscopy. Chemical shift changes for resonances of a number of residues in the vicinity of the active site of the protein have been observed in the presence of urea prior to denaturation. These shifts are similar to those induced in the hen lysozyme spectrum by the specific binding of N-acetylglucosamine (GlcNAc) in site C of the active site cleft, indicating that urea and GlcNAc induce a similar conformational change in the enzyme. This implies that the conformational changes experienced by the enzyme on the binding of GlcNAc oligosaccharides are the consequence of interactions, possibly hydrogen bonding, involving the N-acetyl group of the sugar residue bound in site C, rather than the result of contacts between the protein and the pyranose rings of the oligosaccharides. This suggests that hen lysozyme employs an induced fit type mechanism to discriminate for N-acetylated saccharides as substrates.

Acetylglucosamine↗
PubMed1992

A study of D52S hen lysozyme-GlcNAc oligosaccharide complexes by NMR spectroscopy and electrospray mass spectrometry.

The production of a mutant hen lysozyme is described in which Asp-52, one of the catalytically important residues, is replaced by Ser. The mutant enzyme has very low catalytic activity but NMR studies show that its structure is closely similar to that of the wild-type protein. NMR experiments also show that well defined complexes are formed with GlcNAc4 and GlcNAc6 bound in the active site of the mutant enzyme. These complexes have been examined using electrospray mass spectrometry (ESMS). The most intense peaks arise from the uncomplexed protein indicating that dissociation takes place in the mass spectrometer under the conditions used here. Peaks from minor species corresponding to complexes between the protein and the oligosaccharides are, however, also observed. The possibility that the latter arise from novel covalent enzyme-saccharide complexes is discussed.

Acetylglucosamine↗