PDBsum entry 2k3b

Structural protein

PDB id: 2k3b

Contents

Protein chain

59 a.a. *

* Residue conservation analysis

PDB id:

2k3b

Name:

Structural protein

Title:

Seeing the invisible: structures of excited protein states by relaxation dispersion nmr

Structure:

Actin-binding protein. Chain: a. Fragment: sh3 domain. Engineered: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Gene: abp1. Expressed in: escherichia coli.

NMR struc:

10 models

Authors:

P.Vallurupalli,F.D.Hansen,L.E.Kay

Key ref:

P.Vallurupalli et al. (2008). Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci U S A, 105, 11766-11771. PubMed id: 18701719 DOI: 10.1073/pnas.0804221105

Date:

30-Apr-08 Release date: 29-Jul-08

Protein chain

P15891  (ABP1_YEAST) -  Actin-binding protein from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 592 a.a.

Struc: 59 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1073/pnas.0804221105

Proc Natl Acad Sci U S A 105:11766-11771 (2008)

PubMed id: 18701719

Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy.

P.Vallurupalli, D.F.Hansen, L.E.Kay.

ABSTRACT

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of (15)N, (1)HN, (13)C(alpha), and (13)CO chemical shifts, along with (1)HN-(15)N, (1)H(alpha)-(13)C(alpha), and (1)HN-(13)CO residual dipolar couplings and (13)CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with (1)HN-(15)N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.

Selected figure(s)

Figure 1.
Probing chemical shifts and residual anisotropic interactions in the invisible, excited state. (A) Stick model of a polypeptide chain fragment highlighting the dipolar (^1HN-^15N, ^1H^α-^13C^α, ^1HN-^13CO) and chemical shift anisotropy (^13CO) interactions of the invisible, excited state that are probed (arrows), as well as the chemical shifts (^1HN, ^15N, ^13C^α and ^13CO) that are measured (balls) by the relaxation dispersion NMR experiments that were recorded for the present work. (B–F) Typical relaxation dispersion profiles recorded on samples of ^15N,^2H- (B, C, and F), ^13C^α- (D), or ^15N,^13CO,^2H- (E) labeled Abp1p SH3 domain and substoichiometric amounts of Ark1p peptide (≈5–10% by mole fraction, depending on the sample) at static magnetic field strengths of 500 and 800 MHz (red and blue points, respectively), 25°C, along with global fits of the data to a model of two-site chemical exchange (solid lines).

Figure 3.
Solution structure of the invisible, Ark1p-peptide bound conformation of the Abp1p SH3 domain. (A) Ensemble of 10 starting structures generated from high temperature molecular dynamics of the apo-Abp1p SH3 domain x-ray structure (22), as described in the text. Regions in gray are fixed to the x-ray structure, because Δϖ[RMS] ≈ 0. The pair-wise rmsd values of the backbone C^α, CO and N atoms of regions 1 (red), 2 (blue) and 3 (green) are 5.8 ± 1.6, 3.7 ± 1.0, 2.6 ± 0.7 Å, respectively (10.4 ± 1.6, 8.6 ± 1.2, 3.8 ± 0.6 Å with respect to the apo-Abp1p1 SH3 domain x-ray coordinates). (B) Ensemble of the 10 lowest energy structures generated using (φ, ψ) restraints exclusively, as described in the text. Pair-wise rmsd values of regions 1, 2 and 3 are 3.1 ± 1.4, 3.8 ± 2.4 and 1.2 ± 0.7 Å, respectively. (C) As in B, but including restraints from residual anisotropic interactions as measured using a single alignment media (Pf1). The rmsd values of 0.39 ± 0.12, 0.30 ± 0.11 and 0.20 ± 0.06 Å are calculated for regions 1–3 (0.47 ± 0.10, 0.76 ± 0.14 and 0.53 ± 0.07 Å to the reference structure of the bound form). Inset, ribbon diagram of the apo-Abp1p SH3 domain x-ray structure; all conformers in the figure are in the same orientation. All of the rmsd values reported are calculated by superimposing the “fixed” regions (gray in Fig. 2C); the fixed regions are not included in the computation.

Figures were selected by an automated process.