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I Pelczer

Publications and source records attributed to I Pelczer.

9 recordsLinked to original sources
PubMed1999

Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor.

A mutant tryptophan repressor (TrpR) protein containing the substitution of phenylalanine for leucine 75 has been isolated following a genetic screen for temperature-sensitive mutations. Two-dimensional (2D) 1H NMR spectra indicate an overall very similar fold for the purified mutant and wild-type proteins. Circular dichroism spectropolarimetry indicates an increased helix content relative to the wild-type protein, and a slightly higher urea denaturation midpoint for the mutant protein, although there is no difference in thermal stability. Fluorescence spectra indicate a more buried environment for one or both tryptophan residues in the mutant protein. The rate of proton-deuterium exchange-out for the resolved indole ring protons of the two tryptophan residues was quantified from NMR spectra of mutant and wild-type proteins and found to be approximately 50% faster in the wild-type protein. The mutant protein binds the corepressor l-tryptophan (l-Trp) approximately ten times more weakly than does the wild-type protein, but in l-Trp excess its DNA-binding affinity is only two to fivefold weaker. Taken together the results imply that, despite its conservative chemical character and surface location at the C terminus of helix one in the helix-turn-helix DNA recognition motif, this mutational change confers long-range effects on the dynamics of the protein's secondary and tertiary structure without substantially altering its fold, and with relatively minor effects on protein function.

Amides↗
PubMed1998

Three-dimensional folding of an RNA hairpin required for packaging HIV-1.

An NMR-based structure is presented for a 20 mer hairpin model of the SL3 stem-loop from the HIV-1 packaging signal. The stem has an A-family structure. However, the GGAG tetraloop appears to be flexible with the second (G10) and fourth (G12) bases extruded from the normal stacking arrangement. The A-base (A11) occupies a cavity large enough for it to jump rapidly between stacking upon G9 (in the loop) and G13 (from the base-pair adjacent to the loop). The H-bonding loci of G10, A11, and G12 are unoccupied in the free RNA structure. The loop should be easily adaptable to binding by the HIV-1 nucleocapsid protein or loop receptors.

Base Pairing↗
PubMed1998

The solution structure of the DNA-binding domain of Skn-1.

Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early in the development of Caenorhabditis elegans. It has been reported that the DNA-binding domain is a molten globule and that the structure cannot be defined because there are no long-range nuclear Overhauser effects (NOEs). Working with short Skn domain fragments and using 13C-labeled proteins, we have been able to identify 28 long-range NOEs that establish a tertiary fold for the Skn domain. The internal region of the Skn domain consists of three stable helices and one conformationally labile helix organized into a nascent helix-turn-helix-turn-helix-turn-helix motif. The N and C termini of the Skn domain are unstructured and emerge from the same end of the folded domain. This structure is consistent with biochemical data on binding of the Skn domain to DNA, which shows that the N and C termini bind in the adjacent minor and major grooves from the same face of the DNA helix. The NMR solution structure of the Skn domain should be useful for developing a complete understanding of the DNA recognition event, including any conformational changes that take place upon binding.

Amino Acid Sequence↗
PubMed1998

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: application of the 1H 3D-NOE/2QC experiment.

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the 1H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5'/H5" for RNA plus H2'/H2" for DNA), as well as for H5/H6, for H3'/H4' in sugars with substantial populations of the N-pucker, for H1'/H2' for S-puckered sugars, and usually for H2'/H3'. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.

Capsid↗
PubMed1997

Skn-1: evidence for a bipartite recognition helix in DNA binding.

Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early in the development of Caenorhabditis elegans. This transcription factor contains a basic region, but it binds to DNA as a monomer. Because other transcription factors containing basic regions bind as dimers, this finding implied that Skn represents a new DNA recognition motif. It has been proposed that the basic region helix of Skn is stabilized for binding by tertiary contacts to other parts of the protein. We have tested this proposal by carrying out circular dichroism (CD) and NMR experiments on the Skn domain and five truncated proteins. Our results have shown that the basic region of Skn is unstructured in solution and does not contact other parts of the protein; like other basic region peptides, it folds into a helix only upon binding specifically to DNA. However, there is a stably folded helical module in the Skn domain, and one of the helices in this module terminates immediately before the start of the basic region. This pre-organized helix contains a surface rich in basic amino acids, and we propose that this helix contacts the DNA distal to the basic region proper, providing an extra long helical recognition surface which helps to stabilize monomeric binding. Homology between the Skn domain and several basic-region leucine zipper (bZIP) domains raises the possibility that the affinity and perhaps the specificity of DNA binding by bZIP proteins can be modulated by incorporating a stably folded helical segment that contacts the DNA just below the basic region proper.

Amino Acid Sequence↗
PubMed1996

Improved proton assignment for DNA by application of aliasing and dispersive-absorptive phasing to two-quantum COSY spectra.

A unique combination of aliasing and dispersive-absorptive (DA) phasing of two-quantum correlated spectroscopy (2 Q-COSY) NMR data is shown to enhance proton chemical-shift assignments in DNA oligonucleotides by (i) reducing the time necessary for acquiring NMR data or, alternatively, improving the spectral resolution in a given time, (ii) reducing the number of spectra necessary for NMR data processing and analysis, and (iii) increasing the complexity of oligonucleotide sequences and structures which are accessible to 2D NMR analysis. Aliasing allows a reduction in the size of the acquired data without significant risk of losing information. Phasing the 2Q-COSY dispersive in the F2 dimension reduces the primary antiphase doublet into a pseudo-singlet and increases the apparent signal-to-noise. A single 2Q-COSY spectrum can provide an amount of chemical-shift information comparable to that from a series of COSY, relayed-COSY, and/or spin-lock COSY spectra optimized for various coupling constants. The low signal-to-noise inherent in the most popular two-quantum-filtered correlated spectroscopy (2QF-COSY) of samples with naturally broad lines is largely avoided due to less cancellation. There is no diagonal in a 2Q-COSY which can obscure correlations between protons which are nearly isochronous. As an example of this efficient application, the assignment of 139 of the 143 proton resonances from a single 2Q-COSY and a 2D-NOE spectrum of the DNA hexadecamer [d(AAATATAGCTATATTT)]2 is demonstrated.

Base Sequence↗
PubMed1995

Proton NMR and structural features of a 24-nucleotide RNA hairpin.

The three-dimensional conformation of a 24-nucleotide variant of the RNA binding sequence for the coat protein of bacteriophage R17 has been analyzed using NMR, molecular dynamics, and energy minimization. The imino proton spectrum is consistent with base pairing requirements for coat protein binding known from biochemical studies. All 185 of the nonexchangeable protons were assigned using a variety of homonuclear 2D and 3D NMR methods. Measurements of nuclear Overhauser enhancements and two-quantum correlations were made at 500 MHz. New procedures were developed to characterize as many resonances as possible, including deconvolution and path analysis methods. An average of 21 distance constraints per residue were used in molecular dynamics calculations to obtain preliminary folded structures for residues 3-21. The unpaired A8 residue is stacked in the stem, and the entire region from G7 to C15 in the upper stem and loop appears to be flexible. Several of these residues have a large fraction of S-puckered ribose rings, rather than the N-forms characteristic of RNA duplexes. There is considerable variation in the low-energy loop conformations that satisfy the distance constraints at this preliminary level of refinement. The Shine-Dalgarno ribosome binding site is exposed, and only two apparently weak base pairs would have to break for the 16S ribosomal RNA to bind and the ribosome to initiate translation of the replicase gene. Although the loop form must be regarded as tentative, the known interaction sites with the coat protein are easily accessible from the major groove side of the loop.

Base Sequence↗
PubMed1991

Solid-state and solution conformation of 3'-amino-3'-deoxythymidine, precursor to a noncompetitive inhibitor of HIV-1 reverse transcriptase.

The recent finding that 3'-amino-3'-deoxythymidine 5'-triphosphate is a noncompetitive inhibitor of the HIV-1 reverse transcriptase (Kedar, P.S.; et al. Biochemistry 1990, 29, 3603-3611), prompted an investigation of the conformation of 3'-amino-3'-deoxythymidine. An X-ray diffraction study has revealed that the glycosidic torsion angle of the nucleoside is in the less common syn region and this solid-state geometry is stabilized by a three-dimensional network of self-associated hydrogen-bonded molecules. On the other hand, the aqueous solution conformation, as determined by 1H NMR, places the glycosidic torsion angle in the more usual anti region with the sugar in an equilibrium between C3'-endo and C2'-endo puckering. The energy barrier between the solid-state and solution conformation is relatively low as was demonstrated by the MM2 calculations.

Chemical Phenomena↗