Observing structure,function and assembly of single proteins by AFM

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.     

Inhibition of the cell cycle with chemical inhibitors: a targeted approach

Genetic links between deregulation of the cell cycle and cancer are well established. There have been significant recent developments both in our understanding of the molecular mechanisms that control cell cycle progression and in methods for protein structure determination at atomic resolution. These advances have allowed the rational design of small molecules that modulate the cell cycle by competing for sites of protein-protein or protein-ATP interactions. There is considerable optimism that these compounds, a selection of which are here reviewed, will become clinically significant drugs.     

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.     

AFM for structure and dynamics of biomembranes

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.     

AFM for structure and dynamics of biomembranes

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.     

Surface plasmon resonance protein sensor using Vroman effect

We report a new surface plasmon resonance (SPR) protein sensor using the Vroman effect for real-time, sensitive and selective detection of protein. The sensor relies on the competitive nature of protein adsorption onto the surface, directly depending upon protein's molecular weight. The sensor uses SPR for highly sensitive biomolecular interactions detection and the Vroman effect for highly selective detection. By using the Vroman effect we bypass having to rely on bio-receptors and their attachment to transducers, a process known to be complex and time-consuming. The protein sensor is microfabricated to perform real-time protein detection using four different proteins including aprotinin (0.65kDa), lysozyme (14.7kDa), streptavidine (53kDa), and isolectin (114kDa) on three different surfaces, namely a bare-gold surface and two others modified by OH- and COOH-terminated self-assembled monolayer (SAM). The real-time adsorption and displacement of the proteins are observed by SPR and evaluated using an atomic force microscope (AFM). The sensor can distinguish proteins of at least 14.05kDa in molecular weight and demonstrate a very low false positive rate. The protein detector can be integrated with microfluidic systems to provide extremely sensitive and selective analytical capability.     

Neutron protein crystallography: current status and a brighter future

Hydrogen atoms are rarely seen in X-ray protein crystal structures, but are readily visualized by neutron crystallography, even at typical (1.5-2.5A) resolutions. Recent advances in neutron beamlines and deuterium labeling technologies have dramatically extended the scale and range of structures studied. High-quality neutron data can be collected to near atomic resolution ( approximately 1.5-2.5A) for proteins of 50-175kDa molecular weight, from perdeuterated samples, from crystals with volumes of 0.1mm(3) and at cryogenic temperatures (15K). These structures are providing unique and complementary insights into hydrogen-bonding interactions, protonation states, catalytic mechanisms and hydration states of biological structures that are not available from X-ray analysis alone. The new generation of spallation neutron sources promises further 10-50-fold gains in performance.     

Structure and function of apolipoprotein A-I and high-density lipoprotein

Structural biology and molecular modeling have provided intriguing insights into the atomic details of the lipid-associated structure of the major protein component of HDL, apo A-I. For the first time, an atomic resolution map is available for future studies of the molecular interactions of HDL in such biological processes as ABC1-regulated HDL assembly, LCAT activation, receptor binding, reverse lipid transport and HDL heterogeneity. Within the context of this paradigm, the current review summarizes the state of HDL research.     

Structure, function and dynamics of nuclear subcompartments

The nucleus contains a plethora of different dynamic structures involved in the regulation and catalysis of nucleic acid metabolism and function. Over the past decades countless factors, molecular structures, interactions and posttranslational modifications have been described in this context. On the one side of the size scale X-ray crystallography delivers static snapshots of biomolecules at atomic resolution and on the other side light microscopy allows insights into complex structures of living cells and tissues in real time but poor resolution. Recent advances in light and electron microscopy are starting to close the temporal and spatial resolution gap from the atomic up to the cellular level. Old challenges and new insights are illustrated with examples of DNA replication and nuclear protein dynamics.     

Single-molecule studies of membrane proteins

Characterizing membrane proteins with single-molecule techniques provides structural and functional insights that cannot be obtained with conventional approaches. Recent studies show that atomic force microscopy (AFM) in the context of a 'lab on a tip' enables the measurement of multiple parameters of membrane proteins. This multifunctional tool can be applied to probe the oligomeric states and conformational changes of membrane protein assemblies in their native environment. The ability to determine diverse properties at high spatial resolution facilitates the mapping of structural flexibilities, electrostatic potentials and electric currents. By using the AFM tip as tweezer, it is possible to characterize unfolding and refolding pathways of single proteins and the location of their molecular interactions. These interactions dictate the stability of the protein and might be modulated by ligands that alter the protein's functional state.     

In-cell NMR for protein-protein interactions (STINT-NMR)

We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra.     

The atomic structure of rice dwarf virus reveals the self-assembly mechanism of component proteins

Rice dwarf virus (RDV), the causal agent of rice dwarf disease, is a member of the genus Phytoreovirus in the family Reoviridae. RDV is a double-shelled virus with a molecular mass of approximately 70 million Dalton. This virus is widely prevalent and is one of the viruses that cause the most economic damage in many Asian countries. The atomic structure of RDV was determined at 3.5 A resolution by X-ray crystallography. The double-shelled structure consists of two different proteins, the core protein P3 and the outer shell protein P8. The atomic structure shows structural and electrostatic complementarities between both homologous (P3-P3 and P8-P8) and heterologous (P3-P8) interactions, as well as overall conformational changes found in P3-P3 dimer caused by the insertion of amino-terminal loop regions of one of the P3 protein into the other. These interactions suggest how the 900 protein components are built into a higher-ordered virus core structure.     

Structural biology of cellular machines

Multi-component macromolecular machines contribute to all essential biological processes, from cell motility and signal transduction to information storage and processing. Structural analysis of assemblies at atomic resolution is emerging as the field of structural cell biology. Several recent studies, including those focused on the ribosome, the acrosomal bundle and bacterial flagella, have demonstrated the ability of a hybrid approach that combines imaging, crystallography and computational tools to generate testable atomic models of fundamental biological machines. A complete understanding of cellular and systems biology will require the detailed structural understanding of hundreds of biological machines. The realization of this goal demands a concerted effort to develop and apply new strategies for the systematic identification, isolation, structural characterization and mechanistic analysis of multi-component assemblies at all resolution ranges. The establishment of a database describing the structural and dynamic properties of protein assemblies will provide novel opportunities to define the molecular and atomic mechanisms controlling overall cell physiology.     

Composite structural motifs of binding sites for delineating biological functions of proteins

Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures.     

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.     

Atomic force microscopy and spectroscopy of native membrane proteins

Membrane proteins comprise 30% of the proteome of higher organisms. They mediate energy conversion, signal transduction, solute transport and secretion. Their native environment is a bilayer in a physiological buffer solution, hence their structure and function are preferably assessed in this environment. The surface structure of single membrane proteins can be determined in buffer solutions by atomic force microscopy (AFM) at a lateral resolution of less than 1 nm and a vertical resolution of 0.1-0.2 nm. Moreover, single proteins can be directly addressed, stuck to the AFM stylus and subsequently unfolded, revealing the molecular interactions of the protein studied. The examples discussed here illustrate the power of AFM in the structural analysis of membrane proteins in a native environment.     

The CHAIN program: forging evolutionary links to underlying mechanisms

Proteins evolve new functions by modifying and extending the molecular machinery of an ancestral protein. Such changes show up as divergent sequence patterns, which are conserved in descendent proteins that maintain the divergent function. After multiply-aligning a set of input sequences, the CHAIN program partitions the sequences into two functionally divergent groups and then outputs an alignment that is annotated to reveal the selective pressures imposed on divergent residue positions. If atomic coordinates are also provided, hydrogen bonds and other atomic interactions associated with various categories of divergent residues are graphically displayed. Such analyses establish links between protein evolutionary divergence and functionally crucial atomic features and, as a result, can suggest plausible molecular mechanisms for experimental testing. This is illustrated here by its application to bacterial clamp-loader ATPases.     

Determining Peptide Partitioning Properties via Computer Simulation

The transfer of polypeptide segments into lipid bilayers to form transmembrane helices represents the crucial first step in cellular membrane protein folding and assembly. This process is driven by complex and poorly understood atomic interactions of peptides with the lipid bilayer environment. The lack of suitable experimental techniques that can resolve these processes both at atomic resolution and nanosecond timescales has spurred the development of computational techniques. In this review, we summarize the significant progress achieved in the last few years in elucidating the partitioning of peptides into lipid bilayer membranes using atomic detail molecular dynamics simulations. Indeed, partitioning simulations can now provide a wealth of structural and dynamic information. Furthermore, we show that peptide-induced bilayer distortions, insertion pathways, transfer free energies, and kinetic insertion barriers are now accurate enough to complement experiments. Further advances in simulation methods and force field parameter accuracy promise to turn molecular dynamics simulations into a powerful tool for investigating a wide range of membrane active peptide phenomena.