Activating region of HIV-1 Tat protein: vacuum UV circular dichroism and energy minimization

Abstract

Tat protein is a trans-acting transcriptional activator of the human immunodeficiency virus type 1 and is essential for viral transcription. By homology with other transcriptional activators, Tat is expected to possess a nucleic acid binding region and a separate adjacent activating region. In order to localize the activating region of Tat, we have synthesized the sequences 2-23 and 38-60 of the protein. These two peptides contain the two candidates for the activating regions proposed from mutation experiments in previous studies: sequence 1-13 and sequence 38-45. The argument advanced to justify the location of the activating region within the sequence 1-13 was the periodicity of acidic, polar, and hydrophobic residues consistent with that of an amphipathic alpha-helix, similar to the activating region of many eukaryotic transcriptional activators. We have monitored by vacuum UV circular dichroism the ability of each peptide to adopt an alpha-helical conformation under conditions that strongly favor the formation of secondary structures. Only peptide 38-60 adopts an alpha-helical conformation in these conditions, in keeping with Chou-Fasman prediction. Energy minimization and molecular dynamics were carried out for several possible conformations of sequences 1-14 and 38-60. Our results indicate that only the sequence 38-45 is able to form an alpha-helix with amphipathic characteristics.

A Peptide from Autoantigen La Blocks Poliovirus and Hepatitis C Virus Cap-Independent Translation and Reveals a Single Tyrosine Critical for La RNA Binding and Translation Stimulation

“…These PTDs are similar to LAP in that they rapidly and efficiently enter cells at 37 and 4°C. There are no sequence homologies between LAP and Tat or ANTP PTDs, but protein structure-predicting algorithms strongly suggest that the Tat PTD can adopt an ␣-helix conformation, and the ANTP PTD corresponds to the third ␣-helix of antennapedia homeodomain (19,46). As mentioned above, LAP has a predicted ␣-helix domain, and disruption of this domain as in mutants 761 and 762 dramatically decreases the transduction of LAP (Table 2).…”

Conformational analysis of the Campylobacter jejuni porin

“…Amino acid analyses were performed on a Beckman model 6300 analyzer as previously described (29). Protein samples were hydrolyzed in 6 M HCl-1% phenol for 20 h at 110ЊC.…”

Circular dichroism and molecular modeling yield a structure for the complex of human immunodeficiency virus type 1 trans-activation response RNA and the binding region of Tat, the trans-acting transcriptional activator.

“…One model proposes that the imino nitrogens of the guanidino side chain form specific networks of hydrogen bonds to bridge adjacent phosphodiesters, linking nucleotides G21 to A22 and A22 to U23 in the TAR sequence (12). Recent molecular models of the arginine-rich region of Tat resulting from CD studies suggest that this basic region is an extended structure (15) or is unstructured in the absence of RNA (10). Upon binding to the TAR RNA, however, the peptide may become partially or fully structured and induce a conformational change in the RNA (10).…”

“…§1734 solely to indicate this fact. 9735 320 nm to 178 nm with a VUV-CD spectrophotometer as described (15). Measurements were performed at 20°C for the peptides alone and 5°C for TAR and its peptide complexes (5°C was required to stabilize the complexes).…”

“…Initial models for the TAR RNA alone and TAR complexed with the Tat peptides were constructed by using the IN-SIGHTII program from Biosym Technologies, similar to previous work (15). Each model was subjected to energy minimization, followed by 1 ps of molecular dynamics at 500 K after equilibration, and then final energy minimization to a maximum derivative of 1.0 kcal per step by using the program DISCOVER from Biosym Technologies, with the AMBER (20) forcefields, running on a Silicon Graphics 4D30GTX Iris personal workstation.…”