Biomedical subjects

M H Hecht

Publications and source records attributed to M H Hecht.

31 records · Page 2Linked to original sources

PubMed1994

Periplasmic fractionation of Escherichia coli yields recombinant plastocyanin despite the absence of a signal sequence.

Poplar plastocyanin has been expressed in E. coli from a synthetic gene cloned into the T7 expression system. Despite the absence of a signal sequence, large quantities of the recombinant protein were readily obtained by procedures typically used to isolate proteins from the bacterial periplasm. Several different fractionation methods were equally successful. The presence of plastocyanin in these fractions does not reflect wholesale leakage of intracellular proteins, since neither beta-galactosidase activity nor the bulk of Escherichia coli proteins were released by the fractionation. The identity of the overexpressed protein was unequivocally proven to be poplar plastocyanin by N-terminal amino acid sequence analysis and by spectroscopic characterization of the purified blue copper protein.

Amino Acid Sequence↗

PubMed1994

Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing.

Repeated cycles of freezing and thawing are sufficient to separate highly expressed recombinant proteins away from the cellular milieu of E. coli. Freezing and thawing liberates recombinant proteins from the bacterial cytoplasm, but does not release the bulk of endogenous E. coli proteins. Furthermore, protein secretion is not required. Fractionation of overexpressed proteins by freeze/thaw treatment does not depend on the identity of the recombinant protein and has been observed for thirty-five different recombinant proteins expressed in E. coli. These include proteins originally found in plant, animal or microbial sources, as well as several proteins designed de novo. Freezing and thawing typically yields approximately 50% of the recombinant protein in relatively pure form. Thus the freeze/thaw treatment can be utilized as a general method for the isolation of recombinant proteins from E. coli.

Chemical Fractionation↗

PubMed1993

Protein design by binary patterning of polar and nonpolar amino acids.

A general strategy is described for the de novo design of proteins. In this strategy the sequence locations of hydrophobic and hydrophilic residues were specified explicitly, but the precise identities of the side chains were not constrained and varied extensively. This strategy was tested by constructing a large collection of synthetic genes whose protein products were designed to fold into four-helix bundle proteins. Each gene encoded a different amino acid sequence, but all sequences shared the same pattern of polar and nonpolar residues. Characterization of the expressed proteins indicated that most of the designed sequences folded into compact alpha-helical structures. Thus, a simple binary code of polar and nonpolar residues arranged in the appropriate order can drive polypeptide chains to collapse into globular alpha-helical folds.

Amino Acid Sequence↗

PubMed1993

The role of turns in the structure of an alpha-helical protein.

The turns joining segments of secondary structure have been proposed to be key elements in dictating the folded structures of native proteins. An alternative view assumes that turns play a passive role and are merely default structures that occur as a consequence of interactions between antiparallel segments of secondary structure, with chain reversal being dictated by the context surrounding the turn and not by the sequence of the turn itself. The solvent-exposure of turns and their tolerance to evolutionary variance suggests that they may have little or no effect on the formation of native structures. Previous investigations have focused on various types of beta-turns that connect antiparallel beta-strands with comparatively little reported on the structural role of interhelical turns. Here we probe the structural importance of such a turn in an antiparallel 4-helix bundle by randomly substituting an interhelical tripeptide in cytochrome b-562 with many different amino-acid sequences. Thirty-one of the resulting substituted proteins were characterized and all of them were shown to fold into stable, native-like structures. These results suggest that this interhelical turn does not does not play a dominant role in determining the folded structure of this antiparallel 4-helix bundle.

Amino Acid Sequence↗

PubMed1992

Looking at proteins: representations, folding, packing, and design. Biophysical Society National Lecture, 1992.

Looking at proteins is an active process of interpretation and selection, emphasizing some features and deleting others. Multiple representations are needed, for such purposes as showing motions or conveying both the chain connectivity and the three-dimensional shape simultaneously. In studying and comparing protein structures, ideas are suggested about the determinants of tertiary structure and of folding (e.g., that Greek key beta barrels may fold up two strands at a time). The design and synthesis of new proteins "from scratch" provides a route toward the experimental testing of such ideas. It has also been a fruitful new perspective from which to look at structures, requiring such things as statistics on very narrowly defined structural categories and explicit attention to "negative design" criteria that actively block unwanted alternatives (e.g., reverse topology of a helix bundle, or edge-to-edge aggregation of beta sheets). Recently, the field of protein design has produced a rather unexpected general result: apparently we do indeed know enough to successfully design proteins that fold into approximately correct structures, but not enough to design unique, native-like structures. The degree of order varies considerably, but even the best designed material shows multiple conformations by NMR, more similar to a "molten globule" folding intermediate than to a well ordered native tertiary structure. In response to this conclusion, we are now working on systems that test useful questions with approximate structures (such as determining which factors most influence the choice of helix-bundle topology) and also analyzing how natural proteins achieve unique core conformations (e.g., for side chains on the interior side of a beta sheet, illustrated in the kinemages).

Amino Acid Sequence↗

PubMed1990

De novo design, expression, and characterization of Felix: a four-helix bundle protein of native-like sequence.

The protein Felix was designed de novo to fold into an antiparallel four-helix bundle of specific topology. Its sequence of 79 amino acid residues is not homologous to any known protein sequence, but is "native-like" in that it is nonrepetitive and contains 19 of the 20 naturally occurring amino acids. Felix has been expressed from a synthetic gene cloned in Escherichia coli, and the protein has been purified to homogeneity. Physical characterization of the purified protein indicates that Felix (i) is monomeric in solution, (ii) is predominantly alpha-helical, (iii) contains a designed intramolecular disulfide bond linking the first and fourth helices, and (iv) buries its single tryptophan in an apolar environment and probably in close proximity with the disulfide bond. These physical properties rule out several alternative structures and indicate that Felix indeed folds into approximately the designed three-dimensional structure.

Amino Acid Sequence↗

PubMed1986

Stabilization of lambda repressor against thermal denaturation by site-directed Gly----Ala changes in alpha-helix 3.

Oligonucleotide-directed mutagenesis has been used to replace alpha-helical glycines in the N-terminal domain of lambda repressor with alanines. Since alanine is a significantly better helix-forming residue than glycine, these changes were predicted to have a stabilizing effect. We show that the Gly46----Ala substitution, the Gly48----Ala substitution, and the double substitution increase the melting temperature of the N-terminal domain by 3-6 degrees.

Amino Acid Sequence↗

PubMed1985

Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins.

We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.

Amino Acid Sequence↗

PubMed1985

Increasing and decreasing protein stability: effects of revertant substitutions on the thermal denaturation of phage lambda repressor.

The thermal denaturations of five revertant lambda repressors containing single amino acid substitutions in their N-terminal domains have been studied by differential scanning calorimetry. Two substitutions slightly decrease stability, and the remaining three render the protein more stable than wild type. The Gly48----Asn and Gly48----Ser proteins are 4 degrees C more stable than wild type. These two substitutions replace an alpha helical residue, and in each case a poor helix forming residue, glycine, is replaced by a residue with a higher helical propensity. We also present data showing that one revertant, Tyr22----Phe, has reduced operator DNA binding affinity despite its enhanced stability.

Amino Acid Sequence↗

PubMed1984

Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage lambda repressor.

The thermal stabilities of mutant phage lambda repressors that have single amino acid replacements in the NH2-terminal domain have been studied by means of circular dichroism and differential scanning calorimetry. The variations in stability determined by these physical methods correlate with the resistance to proteolysis at various temperatures and can be compared with the temperature-sensitive activity of the mutants in vivo. In general, mutant proteins bearing solvent-exposed substitutions have thermal stabilities identical to wild type, whereas buried substitutions reduce stability. In one case, a single amino acid replacement increases the thermal stability of the repressor.

Amino Acids↗

PubMed1983

Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding.

The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.

Amino Acid Sequence↗

PubMed1983

The lambda and P22 phage repressors.

The lambda cI repressor and the P22 c2 repressor contain two structural domains. In both proteins, the N-terminal domains mediate operator recognition and positive control of transcription, and the C-terminal domains mediate subunit oligomerization and recognition of the recA protein. In some cases, structural, biochemical, and genetic studies implicate particular repressor side chains in these processes.

Amino Acid Sequence↗

PubMed1983

Mutations defining the operator-binding sites of bacteriophage lambda repressor.

We have characterized about 50 different amino acid substitutions in the aminoterminal domain of lambda repressor. Sixteen of these substitutions alter external side chains of the repressor and cause a substantial reduction in the affinity of the mutant repressor for operator DNA. Seven of these mutant repressors were purified and were shown to be stably folded. The strong, external repressor mutations occur near the aminoterminal end of alpha helix 2, throughout alpha helix 3, and in the aminoterminal-arm region of the repressor. These results suggest that these regions of lambda repressor are close to operator DNA in the protein-DNA complex and thus that these regions comprise the DNA-binding sites of the repressor. Our genetic results support and are completely consistent with more-detailed models of the repressor-operator interaction based on model-building (Pabo and Lewis 1982; Lewis et al. this volume) and biochemical studies (Pabo et al. 1982).

Amino Acid Sequence↗