Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.
Cellulosomes הם קומפלקסי multienzyme בדידים בשימוש על ידי קבוצת משנה של חיידקים אנאירוביים ופטריות לעכל מצעי lignocellulosic. הרכבה של האנזימים על חלבון פיגום noncatalytic בבימויו של אינטראקציות בין בני משפחה של זוגות הקולטן ליגנד נלווים הכוללים מודולים cohesin וdockerin אינטראקציה. חזק מאוד מחייב בין cohesin ותוצאות מודולים dockerin בקבועי דיסוציאציה בpicomolar הנמוכה לטווח nanomolar, שעלולה לעכב מדויקות מדידות מחוץ לשיעור בשיטות בתפזורת קונבנציונלית. כוח ספקטרוסקופיה מולקולה בודדת (SMFS) עם מיקרוסקופ הכוח האטומי מודדת את התגובה של ביומולקולות פרט לכוח, וזאת בניגוד לשיטות אחרות מולקולה בודדת מניפולציה (כלומר פינצטה אופטית), היא אופטימלי לחקר אינטראקציות רצפטור ליגנד זיקה גבוהה בגלל היכולת שלו לחקור את עתיר כוח המשטר (> 120 PN). כאן אנו מציגים הפרוטוקול המלא שלנו ללימוד Cellulמכלולי חלבון osomal ברמת המולקולה בודדת. באמצעות הטופולוגיה חלבון נגזרת מcellulosome הילידים, שעבדנו עם האנזים-dockerin ופחמימות מחייבים מודול-cohesin (CBM-cohesin) חלבוני היתוך, כל אחד עם קבוצת תיאול החופשית נגישה בשאריות ציסטאין מהונדסות. אנו מציגים פרוטוקול קיבוע משטח ספציפי לאתר שלנו, יחד עם המדידה שלנו והליך ניתוח הנתונים לקבלת פרמטרים המחייבים מפורטים למתחם הזיקה גבוהה. אנו מדגימים כיצד לכמת את הכוחות יחיד משנה התגלגלות, כוחות קרע מורכבים, הקינטית מחוץ לשיעורים, ורוחב פוטנציאלי של כריכה היטב. היישום המוצלח של שיטות אלה באפיון האינטראקציה cohesin-dockerin אחראית על הרכבה של קומפלקסי cellulolytic multidomain מתואר נוסף.
Cellulosomes are large multienzyme complexes displayed on the surface of anaerobic cellulolytic bacteria (e.g. C. thermocellum) that have evolved to efficiently depolymerize plant cell wall lignocellulose into soluble oligosaccharides1. A central attribute of cellulosomes is the high-affinity cohesin-dockerin interaction. In the most prominent paradigm, a highly conserved 60-75 amino acid type I dockerin module is displayed at the C-terminal end of the various bacterial enzymes. The dockerin module directs assembly of synergistic combinations of enzymes onto the noncatalytic scaffold protein ('scaffoldin'), which comprises a polyprotein of cohesin domains that are specific for the type I dockerin module. At higher levels, cellulosome architecture can become very complex, incorporating alternative cohesin and dockerin pairs (e.g. type II, type III) that anchor the structures to the cell surface and allow for the assembly of branched structures containing multiple scaffoldins2. The various cohesin-dockerin types, despite having related structures, exhibit differential binding specificities suppressing cross reactivity with unintended scaffoldins or components from other cellulosome-producing bacterial species. While bioinformatic approaches have successfully identified thousands of unique cellulosomal components at the genetic level, comparatively few protein structures are known, and the mechanisms at work in cohesin-dockerin specificity determination remains an active area of structural biology research.
Since the invention of the atomic force microscope (AFM) by Binnig et al.3, various AFM operational modes have been developed and continuously improved, including noncontact imaging, oscillation mode imaging4, and single molecule force spectroscopy (SMFS)5,6. SMFS has evolved into a widely used technique to directly probe individual proteins7-11, nucleic acids12-15, and synthetic polymers16-19. In a typical SMFS experiment to investigate receptor-ligand binding20,21, an AFM cantilever tip is modified with one of the binding partners, while a flat glass surface is modified with the complementary binding partner. The modified cantilever is brought into contact with the surface allowing the partners to bind. The base of the cantilever is then withdrawn at constant speed and the force is measured using the optical lever deflection method. The resultant force-distance data traces exhibit sawtooth-like peaks if binding was established. In cases where the binding partners are fused to multiple protein domains, each peak in the force-distance trace can be correlated to the unfolding of a single protein domain or folded subdomain, while the last peak corresponds to rupture of the protein binding interface. The specific positions of the force-resistant elements can be used as a fingerprint to identify the various protein domains of interest. This method can be used to interrogate important amino acids involved in protein folding and stabilization. Many models have been reported in the literature to treat the characteristic force extension behavior observed in SMFS experiments. The most commonly used models include the freely jointed chain (FJC) model22, the worm-like chain (WLC) model18,23-25, and the freely rotating chain (FRC) model25,26.
In our prior work11, we used single-molecule force spectroscopy to investigate the interaction of cohesin and dockerin modules. Here, we present an experimental protocol for glass surface and cantilever functionalization with enzyme-dockerin and CBM-cohesin protein constructs. We also present an AFM-based SMFS protocol including data acquisition and analysis procedures. The described protocol can easily be generalized to other molecular systems, and should prove particularly useful to researchers interested in high-affinity receptor ligand pairs.
To obtain meaningful data from single molecule force spectroscopy experiments, it is crucial to achieve well-defined and reproducible pulling geometries. The protocol used here results in site-specific immobilization of protein complexes in a defined pulling geometry.
The cantilevers used in this study were chosen due to their force sensitivity and high resonance frequency in water. Moreover, the small tip curvature of approximately 10 nm is advantageous for single molecule experiments due to …
The authors have nothing to disclose.
The authors acknowledge funding from a European Research Council advanced grant to Hermann Gaub. Michael A. Nash gratefully acknowledges funding from Society in Science – The Branco Weiss Fellowship program. The authors thank Edward A. Bayer, Yoav Barak, and Daniel B. Fried at the Weizmann Institute of Science for generously providing the proteins used in this study. The authors thank Hermann E. Gaub, Elias M. Puchner, and Stefan W. Stahl for helpful discussions.
3-Aminopropyl dimethyl ethoxysilane | ABCR GmbH | AB110423 | |
5 kDa NHS-PEG-maleimide | Rapp Polymer | 13 5000-65-35 | |
TCEP Disulfide reducing gel | Thermo Scientific, Pierce | 77712 | www.thermoscientific.com/pierce |
Tris(hydroxymethyl)aminomethane | |||
BioLever mini silicon nitride cantilevers | Olympus | BL-AC40TS-C2 | Soft batches |
XYZ Piezoelectric actuators | Physik Instrumente GmbH | ||
Infrared “broad spectrum” IR laser | Superlum | ||
MFP-3D AFM Controller | Asylum Research | ||
Igor Pro 6.31 | Wavemetrics | Data acquisition and analysis | |
Sodium chloride | |||
Calcium chloride | |||
pH Meter | |||
Sodium borate | |||
Tweezers | |||
Cover glasses | Thermo Scientific, Menzel-Gläser | 24 mm diameter, 0.5 mm thickness | |
PTFE sample holder | custom made | ||
Sonicator bath | |||
Ethanol | analytical purity | ||
Sulfuric acid (concentrated) | analytical purity | ||
Hydrogen peroxide (30%) | analytical purity | ||
Orbital shaker | |||
Toluene | analytical purity | ||
Filter paper | |||
Glass slides | |||
Microtubes | |||
Micropipettes | |||
Centrifuge | suitable for microtubes | ||
Rotator | |||
Petri dishes | |||
Beakers | |||
Optical microscope |