Unveiling the nanoscale architectures and dynamics of protein assembly with in situ atomic force microscopy

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter-and intra-molecular interactions.Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes.Furthermore,the principle of protein assembly gives guidelines for new biomimetic materials with potential appli-cations in medicine,energy,and nanotechnology.Atomic force microscopy(AFM)is a powerful tool for investigating protein assembly and interactions across spatial scales(single molecules to cells)and temporal scales(milliseconds to days).It has significantly contributed to understanding nanoscale architectures,inter-and intra-molecular interactions,and regulatory elements that determine protein structures,assemblies,and functions.This review describes recent advancements in elucidating protein assemblies with in situ AFM.We discuss the structures,diffusions,interac-tions,and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging,bimodal imaging,live cell imaging,and machine-learning-enhanced data analysis.These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.

Combination of NMR spectroscopy and X-ray crystallography offers unique advantages for elucidation of the structural basis of protein complex assembly

NMR spectroscopy and X-ray crystallography are two premium methods for determining the atomic structures of macro-biomolecular complexes.Each method has unique strengths and weaknesses.While the two techniques are highly complementary,they have generally been used separately to address the structure and functions of biomolecular complexes.In this review,we emphasize that the combination of NMR spectroscopy and X-ray crystallography offers unique power for elucidating the structures of complicated protein assemblies.We demonstrate,using several recent examples from our own laboratory,that the exquisite sensitivity of NMR spectroscopy in detecting the conformational properties of individual atoms in proteins and their complexes,without any prior knowledge of conformation,is highly valuable for obtaining the high quality crystals necessary for structure determination by X-ray crystallography.Thus NMR spectroscopy,in addition to answering many unique structural biology questions that can be addressed specifically by that technique,can be exceedingly powerful in modern structural biology when combined with other techniques including X-ray crystallography and cryo-electron microscopy.

Recent advances towards single biomolecule level understanding of protein adsorption phenomena unique to nanoscale polymer surfaces with chemical variations

Protein adsorption onto polymer surfaces is a very complex and ubiquitous phenomenon whose integrated process impacts essential applications in our daily lives such as food packaging materials,health devices,diagnostic tools,and medical products.Increasingly,novel polymer materials with greater chemical intricacy and reduced dimensionality are used for various applications involving adsorbed proteins on their surfaces.Hence,the nature of protein-surface interactions to consider is becoming much more complicated than before.A large body of literature exists for protein adsorption.However,most of these investigations have focused on collectively measured,ensemble-averaged protein behaviors that occur on macroscale and chemically unvarying polymer surfaces instead of direct measurements at the single protein or sub-protein level.In addition,interrogations of protein-polymer adsorption boundaries in these studies were typically carried out by indirect methods,whose insights may not be suitably applied for explaining individual protein adsorption processes occurring onto nanostructured,chemically varying polymer surfaces.Therefore,an important gap in our knowledge still exists that needs to be systematically addressed via direct measurement means at the single protein and sub-protein level.Such efforts will require multifaceted experimental and theoretical approaches that can probe multilength scales of protein adsorption,while encompassing both single proteins and their collective ensemble behaviors at the length scale spanning from the nanoscopic all the way to the macroscopic scale.In this review,key research achievements in nanoscale protein adsorption to date will be summarized.Specifically,protein adsorption studies involving polymer surfaces with their defining feature dimensions and associated chemical partitions comparable to the size of individual proteins will be discussed in detail.In this regard,recent works bridging the crucial knowledge gap in protein adsorption will be highlighted.New findings of intriguing protein surface assembly behaviors and adsorption kinetics unique to nanoscale polymer templates will be covered.Single protein and sub-protein level approaches to reveal unique nanoscale protein-polymer surface interactions and protein surface assembly characteristics will be also emphasized.Potential advantages of these research endeavors in laying out fundamentally guided design principles for practical product development will then be discussed.Lastly,important research areas still needed to further narrow the knowledge gap in nanoscale protein adsorption will be identified.

Protein self-assembly: technology and strategy

Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by this, recent chemical and biological stud- ies mainly focus on supramolecular self-assembly of proteins into high ordered architectures, especially the assembly suategy to unravel tile formation and function of protein nanostructures. In this review, we st, mmarize the progress made in the engi- neering of supramolecular protein architectures according to the strategies used to control the orient：ilion and the order of the assembly process. Furthermore, potential applications in biomedical areas of the supramolecular protein nanostructures will also be reviewed.

Nucleus-Encoded Light-Harvesting Chlorophyll a/b Proteins are Imported Normally into Chlorophyll b-Free Chloroplasts of Arabidopsis

Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light- harvesting chlorophyll a/b-binding proteins （LHCPs）. After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO （chlorophyll a oxigenase） which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant （cao-1）. We imported in vitro translated and 35S-labeled precursor proteins of light- harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex （LHC） proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.

Affinity-guided protein conjugation: the trilogy of covalent protein labeling, assembly and inhibition

Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an "affinity-guided covalent conjugation" strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative(which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made "reactive peptides" for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of "affinity-guided covalent conjugation" in various applications.

Structural insights into the functions of Raf1 and Bsd2 in hexadecameric Rubisco assembly

Hexadecameric formI Rubisco,which consisting consists of eight large(RbcL)and eight small(RbcS)subunits,is the most abundant enzyme on earth.Extensive efforts to engineer an improved Rubisco to speed up its catalytic efficiency and ultimately increase agricultural productivity.However,difficulties with correct folding and assembly in foreign hosts or in vitro have hampered the genetic manipulation of hexadecameric Rubisco.In this study,we reconstituted Synechococcus sp.Pcc6301 Rubisco in vitro using the chaperonin system and assembly factors from cyanobacteria and Arabidopsis thaliana(At).Rubisco holoenzyme was produced in the presence of cyanobacterial Rubisco accumulation factor 1(Raf1)alone or both AtRaf1 and bundle-sheath defective-2(AtBsd2)from Arabidopsis.RbcL released from GroEL is assembly capable in the presence of ATP,and AtBsd2 functions downstream of AtRaf1.Cryo-EM structures of RbcL8-AtRaf18,RbcL8-AtRaf14-AtBsd2s,and RbcLs revealed that the interactions between RbcL and AtRaf1 are looser than those between prokaryotic RbcL and Raf1,with AtRaf1 tilting 7°farther away from RbcL.AtBsd2 stabilizes the flexible regions of RbcL,including the N and C termini,the 60s loop,and loop 6.Using these data,combined with previous findings,we propose the possible biogenesis pathways of prokaryotic and eukaryotic Rubisco.

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex, the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.

Magnetogenetics: remote non-invasive magnetic activation of neuronal activity with a magnetoreceptor

Current neuromodulation techniques such as optogenetics and deep-brain stimulation are transforming basic and translational neuroscience. These two neuro- modulation approaches are, however, invasive since surgical implantation of an optical fiber or wire electrode is required. Here, we have invented a non-invasive magnetogenetics that combines the genetic targeting of a mag- netoreceptor with remote magnetic stimulation. The noninvasive activation of neurons was achieved by neuronal expression of an exogenous magnetoreceptor, an iron-sulfur cluster assembly protein 1 （Iscal）. In HEK-293 cells and cultured hippocampal neurons expressing this magnetoreceptor, application of an external magnetic field resulted in membrane depolarization and calcium influx in a reproducible and reversible manner, as indicated by the ultrasensitive fluorescent calcium indicator GCaMP6s.Moreover, the magnetogenetic control of neuronal activity might be dependent on the direction of the magnetic field and exhibits on-response and off-response patterns for the external magnetic field applied. The activation of this magnetoreceptor can depolarize neurons and elicit trains of action potentials, which can be triggered repetitively with a remote magnetic field in whole-cell patch-clamp recording. In transgenic Caenorhabditis elegans expressing this magnetoreceptor in myo-3-specific muscle cells or mec-4- specific neurons, application of the external magnetic field triggered muscle contraction and withdrawal behavior of the worms, indicative of magnet-dependent activation of muscle cells and touch receptor neurons, respectively. The advantages of magnetogenetics over optogenetics are its exclusive non-invasive, deep penetration, long-term continuous dosing, unlimited accessibility, spatial uniformity and relative safety. Like optogenetics that has gone through decade-long improvements, magnetogenetics, with continuous modification and maturation, will reshape the current landscape of neuromodulation toolboxes and will have a broad range of applications to basic and translational neuroscience as well as other biological sciences. We envision a new age of magnetogenetics is coming.

The formation of fibers via complementary base pairing of DNA-conjugated bovine serum albumin

Assembled protein-based substances are emerging and promising classes of materials that provide unique properties for various applications in biotechnology and nanotechnolegy. Self-assembly is an effective way to immobilize protein. In this study, DNAs-conjugated bovine serum albumin （BSA） assembled into fibers via DNA hybridization is demonstrated. The morphology of fibers was observed by optical microscopy, scanning electron microscopy （SEM）, and atomic force microscopy （AFM）, and the assembly mechanism was then analysed and discussed. BSA molecules were first linked by DNA molecule and formed linear chains. These chains then were parallelly linked through additional DNA hybridization. Finally, several BSA chains further assembled into fibers by layering lamellae in a parallel manner. This work perhaps will provide a guide to the immobilization of enzyme, which could be applied to increase its catalytic efficiency in biomedicine and nanotechnology.