Gaetano T. Montelione

Consistent blind protein structure generation from NMR chemical shift data

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the 13Cα, 13Cβ, 13C′, 15N, 1Hα and 1HN NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7–1.8 Å root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination.

Evaluating Protein Structures Determined by Structural Genomics Consortia

Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X‐ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely‐used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283–291), MolProbity (Lovell et al., Proteins 2003;50:437–450), Verify3D (Luthy et al., Nature 1992;356:83–85), ProsaII (Sippl, Proteins 1993;17: 355–362), the PDB validation software, and various structure‐validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness‐of‐fit between structures and experimental data, and knowledge‐based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site‐specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high‐resolution X‐ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X‐ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5‐year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher‐resolution X‐ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this “structure quality score gap” between NMR and X‐ray crystal structures are discussed. Proteins 2007.

Principles for designing ideal protein structures

Unlike random heteropolymers, natural proteins fold into unique ordered structures. Understanding how these are encoded in amino-acid sequences is complicated by energetically unfavourable non-ideal features—for example kinked α-helices, bulged β-strands, strained loops and buried polar groups—that arise in proteins from evolutionary selection for biological function or from neutral drift. Here we describe an approach to designing ideal protein structures stabilized by completely consistent local and non-local interactions. The approach is based on a set of rules relating secondary structure patterns to protein tertiary motifs, which make possible the design of funnel-shaped protein folding energy landscapes leading into the target folded state. Guided by these rules, we designed sequences predicted to fold into ideal protein structures consisting of α-helices, β-strands and minimal loops. Designs for five different topologies were found to be monomeric and very stable and to adopt structures in solution nearly identical to the computational models. These results illuminate how the folding funnels of natural proteins arise and provide the foundation for engineering a new generation of functional proteins free from natural evolution.

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

NMR structure determination for larger proteins using backbone-only data.

Examining the Backbone Determination of tertiary protein structures by nuclear magnetic resonance (NMR) currently relies heavily on side-chain NMR data. The assignment of side-chain atoms is challenging. In addition, proteins larger than 15 kilodaltons (kD) must be deuterated to improve resolution and this eliminates the possibility of measuring long-range interproton distance constraints. Now Raman et al. (p. 1014, published online 4 February) use backbone-only NMR data—chemical shifts, residual dipolar coupling, and backbone amide proton distances—available from highly deuterated proteins to guide conformational searching in the Rosetta structure prediction protocol. Using this new protocol, they were able to generate accurate structures for proteins of up to 25 kD. Protein structures can be determined by using the limited nuclear magnetic resonance information obtainable for larger proteins. Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins.

Protein NMR spectroscopy in structural genomics.

Protein NMR spectroscopy provides an important complement to X-ray crystallography for structural genomics, both for determining three-dimensional protein structures and in characterizing their biochemical and biophysical functions.

Structural basis for suppression of a host antiviral response by influenza A virus

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-β mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3′ end processing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-β pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.

Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment

A suite of reduced-dimensionality 13C,15N,1H-triple-resonance NMR experiments is presented for rapid and complete protein resonance assignment. Even when using short measurement times, these experiments allow one to retain the high spectral resolution required for efficient automated analysis. “Sampling limited” and “sensitivity limited” data collection regimes are defined, respectively, depending on whether the sampling of the indirect dimensions or the sensitivity of a multidimensional NMR experiments per se determines the minimally required measurement time. We show that reduced-dimensionality NMR spectroscopy is a powerful approach to avoid the “sampling limited regime”—i.e., a standard set of ten experiments proposed here allows one to effectively adapt minimal measurement times to sensitivity requirements. This is of particular interest in view of the greatly increased sensitivity of NMR spectrometers equipped with cryogenic probes. As a step toward fully automated analysis, the program autoassign has been extended to provide sequential backbone and 13Cβ resonance assignments from these reduced-dimensionality NMR data.

Automated analysis of NMR assignments and structures for proteins

Recent developments in protein NMR technology have provided spectral data that are highly amenable to analysis by advanced computer software systems. Specific data collection strategies, coupled with these computer programs, allow automated analysis of extensive backbone and sidechain resonance assignments and three-dimensional structures for proteins of 50 to 200 amino acids.

High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli

SummaryAn approach to produce 13C-and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40–200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C6-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C6-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purified IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20–40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.