Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Wiley full text link Wiley Free PMC article
Full text links

Actions

Share

.2006 Apr;15(4):871-83.
doi: 10.1110/ps.051880406. Epub 2006 Mar 7.

Computer-aided NMR assay for detecting natively folded structural domains

Affiliations

Computer-aided NMR assay for detecting natively folded structural domains

Takayuki Hondoh et al. Protein Sci.2006 Apr.

Abstract

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Domain prediction. The expressed domains predicted with PASS are indicated by gray ellipses. The first and last residues, the computed and measured molecular masses, and the computed isoelectric point are indicated for each domain. The trans-membrane regions predicted with ALOM are shown with shaded bars.
Figure 2
Figure 2
(A) Generic purification protocol. All of the domains were purified with the same protocol, without any optimization (for details, see Materials and Methods). The sample volume at each purification stage is shown on theright side of the figure. The protocol was designed so that the entire purification, after the initial loading of the protein to the nickel resin (step 1), can be performed in a single 50-mL sample tube. The fraction containing the protein is indicated on theright side of the figure, in italics. The first step, “Cell pellet,” indicates the resuspension and lysis of theE. coli cells (up to 5 g pellets). “Purified sample” indicates the low-concentration protein purified according to this protocol. “Final NMR sample” indicates the concentrated sample used for NMR measurements or as a stock solution for biochemical tests. The time indicated at theleft of each purification step was measured from the time at which the cell pellet was first resuspended for lysis. Steps 1–5 were carried out in 4 h. The time typically needed for concentrating a well-behaving protein to NMR sample concentrations was 2.5 h, and the HSQC spectra were measured on the same day. The broken arrow between steps 5 and 6 indicates that some samples needed a longer concentration/buffer exchange time. This protocol was first tested with DFF-C, an 83-residue natively folded domain of DFF-45. (B) HSQC spectra of DFF-C, purified according to the rapid purification protocol. (C) Spectrum of the conventional Niresin/HPLC-purified DFF-C, which needed ≥5 d for completion. The two spectra are identical, and the peak assignments of DFF-C indicate that all of the residues are visible on both spectra. Furthermore, no impurities originating from peptide fragments are present, as judged by the absence of minor peaks that would indicate their presence.
Figure 3
Figure 3
HSQC spectra and SDS-PAGE gels of the putative domains. (a) HSQC spectra of the final samples, purified according to the protocol described in Figure 2. Since many spectra contained a sizable fraction of cross-peaks with weak intensities, we divided the cross-peaks into two classes according to their intensities. Peaks with strong and weak intensities are numbered in black and gray italics, respectively. Peaks that are likely to correspond to the indole NH of tryptophan are boxed with a broken line. Peak pairs likely to correspond to side-chains NH are indicated by horizontal lines connecting the two peaks. The spectra were displayed with the NMRview software, with drawing parameters set to: level = 1.0, CLM = 1.3, and NLVIS = 30. (b) The yield at each purification step was monitored by SDS-PAGE. The molecular masses in kilodaltons are indicated on theleft of the gel. The lanes are as follows:M, molecular mass marker;C (step 1), cell lysate;S, supernatant;P, precipitate;Ft (step 2), flow through of the nickel resin;W, wash of the nickel resin,Ni, the fraction bound to the nickel resin,Tc (step 3), nickel resin slurry after thrombin digestion,Te (step 5), eluted fraction after thrombin digestion; andFi (step 6), final sample. From a total volume of 50 mL, a 7.8 μL aliquot was loaded in laneC. Equivalent amounts were loaded in lanesS toFt, and double amounts were loaded in lanesW toFi. The black arrow on theright side of the gel indicates the band position of the final purified sample. The numbers at thebottom of the gel indicate the estimated quantity of protein at each step in milligrams (upper line) and in percentages (bottom line). (c) Same HSQC spectra as ina recorded after an overnight incubation at 308K. All of the spectra measured after the incubation period remained unchanged, except for that of 1347(1–119). In the spectrum of 1347(1–119) measured after the overnight incubation, some minor peaks as well as broad peaks collapsed in the spectrum center are visible.
Figure 4
Figure 4
Domain stability and boundary refinement using MALDI-TOF: The SDS-PAGE gel represents the results of limited proteolysis performed with a 1/1000 relative trypsin/protein concentration. The incubation times ranged from 0–60 min and are indicated on thetop of the gels. DFF-C is a control for a natively folded 83-residue domain flanked with a few unfolded residues at both its N and C termini. When the digestion produced one or a few fragments [DFF-C-control, 288(31–178), 1600, and 1347], the band identities were determined unambiguously from the molecular masses measured by MALDI-TOF spectroscopy. In these cases, the fragment identity was determined by comparing the experimental molecular mass with computed molecular masses of all possible fragments generated by trypsin digestion (cleavage at all R and K residues). Identification based on the fragment molecular mass was ambiguous for 288(165–431) and His-2205(120–368) because of the large numbers of fragments with similar molecular masses. In these cases, N-terminal amino acid sequencing provided unambiguous band identification. The first and last residues of the fragments are indicated on theright side of the gels, as well as the measured/calculated molecular masses in parentheses.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albala J.S., Franke K., McConnell I.R., Pak K.L., Folta P.A., Rubinfeld B., Davies A.H., Lennon G.G., Clark R., BertoneP.et al. 2000. From genes to proteins: High-throughput expression and purification of the human proteome J. Cell. Biochem. 80: 187–191. - PubMed
    1. Bateman A., Coin L., Durbin R., Finn R.D., Hollich V., Griffiths-Jones S., Khanna A., Marshall M., Moxon S., SonnhammerE.L.et al. 2004. The Pfam protein families database Nucleic Acids Res. 32: D138–D141. - PMC - PubMed
    1. Brenner S.E. 2000. Target selection for structural genomics Nat. Struct. Biol. 7:Suppl, 967–969. - PubMed
    1. 2001. A tour of structural genomics Nat. Rev. Genet. 2: 801–809 ——. - PubMed
    1. Burley S.K., Almo S.C., Bonanno J.B., Capel M., Chance M.R., Gaas-terland T., Lin D., Sali A., Studier F.W., Swaminathan S. 1999. Structural genomics: Beyond the human genome project Nat. Genet. 23: 151–157. - PubMed

Publication types

MeSH terms

LinkOut - more resources

Full text links
Wiley full text link Wiley Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp