Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs.

The objective of this study was to determine whether the rates of deamidation of Asn8 in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu27]GRF(1-32)NH2 (both having Gly at position 15), [Ala15Leu27]bGRF(1-32)NH2 and [Pro15Leu27]bGRF(1-32)NH2 were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro15Leu27 bGRF(1-32)NH2, [Leu27]hGRF(1-32)NH2 and Ala15Leu27 bGRF(1-32)NH2, respectively. 2D NMR studies done in the presence of 40% CD3OH indicated more helical structure for the Ala15 analog as compared to [Leu27]hGRF(1-32)NH2. In both these peptides Asn8 was included in the helical region. In contrast, the lack of conformational information for the Pro15 analog indicated little helical structure around Asn8. The peptides' deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro15 bGRF analog (t1/2 = 10.78 h) deamidated 1.5 and 2 times faster than its Gly15 (t1/2 = 15.74 h) and Ala15 (t1/2 = 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn8 in GRF makes this residue less prone to deamidation.

Solution conformation of Leu27 hGRF(1-32)NH2 and its deamidation products by 2D NMR.

The solution structure and helical content of a human growth hormone releasing factor analog, Leu27 hGRF(1-32)NH2 (hGRF), and its deamidation products Asp8 Leu27 hGRF(1-32)NH2 and isoAsp8 Leu27 hGRF(1-32)NH2, were determined by CD and 2D NMR. Chemical-shift assignments of 1H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn8, Asp8 and isoAsp8 hGRF analogs to have significant alpha-helical character. However, the beta-linked isoAsp8 analog did not retain helical structure in the N-terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was approximately 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn8, Asp8 and isoAsp8 hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N-terminal region for the isoAsp8 analog when compared with the other two analogs. In the Asn8 and Asp8 hGRF analogs, the helix began at Asp3 or Ala4, while the isoAsp8 analog helix was disrupted until Arg11. The higher helicity value for the Asn8 peptide over the isoAsp8 analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn8, Asp8 and isoAsp8 hGRF, respectively.

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).

Transcriptional enhancer related DNA sequences: anomalous 1H NMR NOE crosspeaks.

A dynamic heterogeneity which correlates with the function of the operator DNA in the lactose operon of E. coli. was previously observed (1) as a local minimum in the thymine imino proton T1 centered at a GTG/C-CAC sequence. Since this triplet occurs frequently in DNA regulatory regions, it was proposed that these sequences may be part of a structural element for specific protein interaction. We examine here three additional biologically significant 17 base pair duplexes containing GTG/CAC triplets: (1) a sequence from the mouse heavy chain immunoglobulin enhancer, (2) a sequence from the critical core of the Simian Virus 40 (SV40) enhancer, and (3) a sequence from pBR322 plasmid used as control for experiments with the SV40 DNA sequences. The 1H NMR resonance assignment for nearly all the nonexchangeable protons for both eukaryotic enhancer duplexes with the exception of the H5'/H5" protons was accomplished to use for structural analysis of these duplexes. The data presented show several NOE's associated with the GTG/CAC triplets which suggest structural variation from uniform B-DNA. In addition, anomalous broad crosspeaks for the fixed thymine methyl to its own H6 proton in combination with the imino proton kinetics associated with these triplets reinforces the original observation of a sequence dependent dynamic variation.