Recent controversies associated with the structure of the M2 protein from influenza A virus and the binding site of drug molecules amantadine and rimantadine motivated the comparison here of the drug binding to three viral porins including the M2 proteins from influenza A and B as well as the viral protein ‘u’ from HIV-1. While the M2 protein from influenza B does not normally bind amantadine, chimeras with the M2 protein from influenza A show blockage by amantadine. Similarly, Vpu does not normally bind rimantadine, but the single site mutation A18H converts a non-specific channel to a selective proton channel that is sensitive to rimantadine. The comparison of structures and amino acid sequences shows that the membrane protein sample environment can have a significant influence on the structural result. While a bilayer surface bound amphipathic helix has been characterized for AM2, such a helix may be possible for BM2 although it has evaded structural characterization in detergent micelles. A similar amphipathic helix seems less likely for Vpu. Even though the A18H Vpu mutant forms rimantadine sensitive proton channels, the binding of drug and its influence on the protein structure appears to be very different from that for the M2 proteins. Indeed, drug binding and drug resistance in these viral porins appears to result from a complex set of factors.
M2 protein; Influenza A virus; Influenza B virus; Viral protein u; HIV; PISEMA; Solid-state NMR; Membrane protein
A sinusoidal modulation scheme is described for selective heteronuclear polarization transfer between two dilute spins in double cross polarization magic-angle-spinning nuclear magnetic resonance spectroscopy. During the second N → C cross polarization, the 13C RF amplitude is modulated sinusoidally while the 15N RF amplitude is tangent. This modulation induces an effective spin-lock field in two selective frequency bands in either side of the 13C RF carrier frequency, allowing for simultaneous polarization transfers from 15N to 13C in those two selective frequency bands. It is shown by experiments and simulations that this sinusoidal modulation allows one to selectively polarize from 15N to its covalently bonded 13Cα and 13C′ carbons in neighboring peptide planes simultaneously, which is useful for establishing the back-bone connectivity between two sequential residues in protein structural elucidation. The selectivity and efficiency were experimentally demonstrated on a uniformly 13C,15N-labeled β1 immunoglobulin binding domain of protein G (GB1).
Double cross polarization (DCP); NCA/NCO; Solid-state MAS NMR; Heteronuclear polarization transfer
The feasibility of using solid-state MAS NMR for in situ structural characterization of the LR11 (sorLA) transmembrane domain in native Escherichia coli (E. coli) membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimer's disease. The background signals from E. coli lipids and membrane proteins had only minor effects on LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using 13C PARIS data. These assignments allow comparisons of the secondary structure of LR11 TM in native membrane environments and in commonly used membrane mimics (e.g. micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis, and offers an opportunity to investigate the consequences of membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding on the protein structure.
Protein aggregation; 19F NMR; Unnatural amino acid; SDS Micelle
Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams per liter of macromolecules often interfere with observing the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide 1H exchange. We analyze the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues where stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions need to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.
Despite increased attention, little is known about how the crowded intracellular environment affects basic phenomena like protein diffusion. Here, we use NMR to quantify the rotational and translational diffusion of a 7.4-kDa test protein, chymotrypsin inhibitor 2 (CI2), in solutions of glycerol, synthetic polymers, proteins, and cell lysates. As expected, translational diffusion and rotational diffusion decrease with increasing viscosity. In glycerol, for example, the decrease follows the Stokes-Einstein and Stokes-Einstein-Debye laws. Synthetic polymers cause negative deviation from the Stokes Laws and affect translation more than rotation. Surprisingly, however, protein crowders have the opposite effect, causing positive deviation and reducing rotational diffusion more than translational diffusion. Indeed, bulk proteins severely attenuate the rotational diffusion of CI2 in crowded protein solutions. Similarly, CI2 diffusion in cell lysates is comparable to its diffusion in crowded protein solutions, supporting the biological relevance of the results. The rotational attenuation is independent of the size and total charge of the crowding protein, suggesting that the effect is general. The difference between the behavior of synthetic polymers and protein crowders suggests that synthetic polymers may not be suitable mimics of the intracellular environment. NMR relaxation data reveal that the source of the difference between synthetic polymers and proteins is the presence of weak interactions between the proteins and CI2. In summary, weak but non-specific, non-covalent chemical interactions between proteins appear to fundamentally impact protein diffusion in cells.
In-cell NMR; Macromolecular crowding; Protein diffusion; Weak interactions
Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm.
Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild (~0.5 kcal/mol at 37°C, pH 5.4) stabilization at a volume occupancy of 70%, which approaches the occupancy of closely packing spheres. The lack of change in rotational dynamics indicates the absence of strong crowder-protein interactions.
Our observations are explained by the large size discrepancy between the protein and crowders and by the internal structure of the microgels, which provide interstitial spaces and internal pores where the protein can exist in a dilute solution-like environment. In summary, microgels that interact weakly with proteins do not strongly influence protein dynamics or stability because these large microgels constitute an upper size limit on crowding effects.
Although over expression and 15N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, 15N enrichment alone is insufficient for detecting most globular proteins. Here we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using 19F labeled proteins. Resonances from small (~10 kDa) globular proteins containing the amino acid analog 3-fluoro-tyrosine can be observed in cells, but for larger proteins the 19F resonances are broadened beyond detection. Incorporating the amino acid analog trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using 19F NMR.
19F NMR; in-cell NMR; intrinsically disordered proteins; site specific labeling; protein structure; protein dynamics
Protein protein interaction is the fundamental step of biological signal transduction. Interacting proteins find each other by diffusion. To gain insight into diffusion under the crowded conditions found in cells, we used nuclear magnetic resonance spectroscopy (NMR) to measure the effects of solvent additives on the translational and rotational diffusion of the 7.4 kDa globular protein, chymotrypsin inhibitor 2. The additives were glycerol and the macromolecular crowding agent, polyvinylpyrrolidone (PVP). Both translational diffusion and rotational diffusion decrease with increasing solution viscosity. For glycerol, the decrease obeys the Stokes Einstein and Stokes Einstein Debye laws. Three types of deviation are observed for PVP: the decrease in diffusion with increased viscosity is less than predicted, this negative deviation is greater for rotational diffusion, and the negative deviation increases with increasing PVP molecular weight. We discuss our results in terms of other studies on the effects of macromolecules on globular protein diffusion.
macromolecular crowding; nuclear magnetic resonance; viscosity
Conventional NMR approaches to detect weak protein binding and aggregation are hindered by the increased viscosity brought about by crowding. We describe a simple and reliable NMR method to distinguish viscosity effects from binding and aggregation under crowded conditions.
Almost everything we know about protein biophysics comes from studies on purified proteins in dilute solution. Most proteins, however, operate inside cells where the concentration of macromolecules can be >300 mg per mL. Although reductionism-based approaches have served protein science well for over a century, biochemists now have the tools to study proteins under these more physiologically-relevant conditions. We review a part of this burgeoning post-reductionist landscape by focusing on high-resolution protein NMR spectroscopy, the only method that provides atomic-level information over an entire protein under the crowded conditions found in cells.
High-resolution solid-state NMR spectra of three full-length membrane proteins uniformly aligned in lipid bilayers between glass slides are observed at high magnetic field. The resolution of the specific amino acid labeled samples shows promise for large membrane protein structure determination utilizing aligned samples and shows resonance patterns known as PISA wheels. The tilt angles of the transmembrane helices are extracted from the resonance patterns in PISEMA spectra.
Theory predicts that macromolecular crowding affects protein behavior, but experimental confirmation is scant. Herein, we report the first residue-level interrogation of the effects of macromolecular crowding on protein stability. We observe up to a 100-fold increase in the stability, as measured by the equilibrium constant for folding, for the globular protein chymotrypsin inhibitor 2 (CI2) in concentrations of the cosolute poly(vinylpyrrolidone) (PVP) that mimic the protein concentration in cells. We show that the increased stability is caused by the polymeric nature of PVP and that the degree of stabilization depends on both the location of the individual residue in the protein structure and the PVP concentration. Our data reinforce the assertion that macromolecular crowding stabilizes the protein by destabilizing its unfolded states.
Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.
M2 channel; Influenza A virus; Conformational plasticity; PISEMA; Solid-state NMR; Membrane protein