Advances in NMR structures of integral membrane proteins

Abstract

Integral membrane proteins (IMPs) play a central role in cell communication with the environment. Their structures are essential for our understanding of the molecular mechanisms of signaling and for drug design, yet they remain badly underrepresented in the protein structure databank. Solution NMR is, aside from X-ray crystallography, the major tool in structural biology. Here we review recently reported solution NMR structures of polytopic IMPs and discuss the new approaches, which were developed in the course of these studies to overcome barriers in the field. Advances in cell-free protein expression, combinatorial isotope labeling, resonance assignment, and collection of structural data greatly accelerated IMP structure determination by solution NMR. In addition, novel membrane-mimicking media made possible determination of solution NMR structures of IMPs in a native-like lipid environment.

Figure 1

Recent new solution NMR structures of polytopic IMPs. The TM helices are colored in the following order starting from the N-terminus: green, blue, orange, dark violet, brown, magenta, and red. Non-TM helices are shown in gray. The PDB codes are indicated under the protein names. Figures are prepared using the Molmol program [65].

Figure 2

Flowchart of high-throughput cell free expression screening and cell-free assisted accelerated structure determination.

Figure 3

Protein sequence analysis using combinatorial dual-labeling strategy [20] for accelerated assignment. From a distribution of amino acids in a sequence, represented as a matrix of amino acid pairs (A), MCCL program (http://sbl.salk.edu/combipro) calculates the combinatorial dual-labeling scheme (B) for maximal possible assignment with available amino acids (E). Each type of the amino acid pairs (A) in a protein sequence receives an assignment tag (C) according to the labeling pattern of the peptide bond between the two amino acids in the pair in each sample (0 – no ¹⁵N labeling of the peptide bond; 1 – ¹⁵N-only; 2 – ¹³C and ¹⁵N labeling).

Figure 4

Accelerated assignment using the CDL strategy [21]. (A) The CDL samples are expressed simultaneously in the CF system according to the CDL scheme and solubilized in the same buffer. The ¹H-¹⁵N-TROSY-HSQC and HN(CO) spectra are acquired for each CDL sample. Panel (C) shows superposition of a fragment of ¹H-¹⁵N-HSQC and HN(CO) spectra for each CDL sample. Analysis of the spectra reveals the assignment code (D) for the cross peak in each sample (according to the rules (B): 0 – absent in both spectra; 1 – present only in HSQC; 2 – present in both HSQC and HNCO). Each of ¹H-¹⁵N cross-peaks is assigned by matching the tag of codes derived from the spectra (D) with the code pre-calculated using the CDL scheme (E).