Biomedical subjects

Stephen M Lu

Publications and source records attributed to Stephen M Lu.

5 recordsLinked to original sources

PubMed2006

Functional evolution within a protein superfamily.

The ability to predict and characterize distributions of reactivities over families and even superfamilies of proteins opens the door to an array of analyses regarding functional evolution. In this article, insights into functional evolution in the Kazal inhibitor superfamily are gained by analyzing and comparing predicted association free energy distributions against six serine proteinases, over a number of groups of inhibitors: all possible Kazal inhibitors, natural avian ovomucoid first and third domains, and sets of Kazal inhibitors with statistically weighted combinations of residues. The results indicate that, despite the great hypervariability of residues in the 10 proteinase-binding positions, avian ovomucoid third domains evolved to inhibit enzymes similar to the six enzymes selected, whereas the orthologous first domains are not inhibitors of these enzymes on purpose. Hypervariability arises because of similarity in energetic contribution from multiple residue types; conservation is in terms of functionality, with "good" residues, which make positive or less deleterious contributions to the binding, selected more frequently, and yielding overall the same distributional characteristics. Further analysis of the distributions indicates that while nature did optimize inhibitor strength, the objective may not have been the strongest possible inhibitor against one enzyme but rather an inhibitor that is relatively strong against a number of enzymes.

Amino Acid Sequence↗

PubMed2004

Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability.

The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.

Alanine↗

PubMed2003

Testing of the additivity-based protein sequence to reactivity algorithm.

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.

Algorithms↗

PubMed2002

A de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins.

The generation of antibodies directed toward the surface-exposed regions of a protein using synthetic peptides as immunogens representing surface loops and turns has been widely successful. However, peptides representing alpha-helical regions are typically unstructured in solution and unable to produce antibodies that recognize alpha-helices in native proteins. We describe a de novo designed parallel two-stranded alpha-helical coiled-coil template for immunization to prepare antibodies that recognize alpha-helical protein sequences in the native protein. This template was designed for maximum stability through an Ile/Leu hydrophobic core and an interchain disulfide bridge. Surface-exposed helical residues are inserted into the template and used for immunization to generate polyclonal antibodies. To demonstrate the feasibility of this approach, 15 residues of the yeast transcription factor GCN4 were inserted into this template, and the resultant antibodies were screened for conformational specificity. Peptide antigens that contain the same surface-exposed residues but differ in structure were used as competitors in a competition assay. Direct competition between the capture peptide immobilized on a biosensor chip, the peptide antigens, and the antibodies generated by the template demonstrated that the antibodies were specific for helical structure in the native coiled-coil (synthetic GCN4 residues 250-280). These antibodies were unable to recognize the same inserted sequence in an unstructured analog. The helix-specific antibodies were also able to identify native GCN4 (31.3 kDa) from yeast whole cell extracts.

Amino Acid Sequence↗

PubMedDate not supplied

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template.

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.

3T3 Cells↗