While nucleic acids are the source of information for maintaining life, proteins & nucleic acid systems are the source of functionality. These macromolecules are capable of functioning as autonomous nano-machines carrying specific activities.
Many of the characteristics allowing this are related to their three-dimensional structure, determining their specificity in most cases. However, without the ability to move and change structure, proteins would facilitate the chemistry of maintaining life.
While nowadays we understand the structural aspect of protein systems, their dynamics and how it facilitates their activity is mostly known to us from computational simulations and there are very few experimental techniques allowing measuring these characteristics with high spatial & temporal resolution.
This laboratory is dedicated to the direct measurement of these characteristics of protein systems one molecule at a time, protein-after-protein.
Using these techniques we study the molecular mechanisms leading to bacterial resistance to antibiotic molecules, through structural changes in the bacterial transcription complex. Additionally, we study the mechanisms allowing the transduction of chemical signals through proteins mechanically, and how they assist in transcriptional regulation in bacteria and in signal transduction in the eukaryotic cell.
Integrative structural biology using single-molecule fluorescence spectroscopy
In many cases, the powerful tools of classical structural biology fail to retrieve the structures of proteins in specific conformational states. Nowadays, single-particle cryo-electron microscopy, together with strong classification algorithms, allow identification and characterization of several different structures of the same protein. Nevertheless, the information on the conformational dynamics is lost. Moreover, current capabilities are of identification of species with abundance as low as 20%. How should the structures of lower abundance conformational states be retrieved?
Single-molecule fluorescence spectroscopy allows us to sort different bio-molecular species according to their inter-atomic distances, rotational freedom, binding stocihiometry, and many other parameters that influence fluorescence. Using this sorting capabilities we aim at identifying new conformational states of proteins and their complexes, that have not yet been structurally characterized. Then, we aim at combining the experimental data with structural modelling capabilities to retrieve the 3D structure of these conformational states.
Using this approach, we aim at identifying the different small oligomers that the protein alpha-Synuclein forms, and deciphering the structure of those small oligomers that cause cell toxicity, leading to accumulation of damage in Parkinson's disease. The goal of this study is to use the structure of the toxic alpha-Synuclein small oligomers to rationally-design inhibitors that will minimize the cellular damage linked to Parkinson's Disease.
Additionally, using this approach, we aim at characterizing the structure of the transcription initiation complex in conformational states to which antibiotics do not bind with high affinity. The goal of this study is to use the structure to understand why standard antiobiotic molecules did not strongly inhibit transcription initiation and to rationally-design inhibitors to strongly inhibit transcription by binding to this conformational state of the transcription initiation complex.
Characterization of toxic biomolecular species in Parkinson's Disease
A well-known molecular feature in Parkinson's Disease (PD), is the accumulation of fibrils of the protein alpha-Synuclein in Lewy bodies, inside neurons of the Substantia Nigra. Alpha-Synuclein is an intrinsically-disordered protein (IDP). As is the case with many other IDP's or proteins with a degree of intrinsically disordered regions (IDRs), the disordered, or better put unfolded regions tend to find alternative ways to fold, usually via the coupled binding to another molecule. In some cases, the alternative folding is coupled to binding of the same protein in a process called oligomerization. In some cases, these oligomers reach a finite size, however in others, oligomer sizes keep on growing to form insoluble aggregates, and in some cases ordered fibrils, that are also insoluble.
While fully-grown fibrils of alpha-Synuclein are a main characteristic of PD, specific small-sized oligomers have the capability to bind to membrames and form pores, which can explain the basis for toxicity in the damage accumulation linked to PD. These specific oligomers are not abundant and also transient in ther nature, which can explain why damage accumulation in PD is so slow.
The current view of the process of fibril formation is that of a nucleation process, in which fibrils are formed from an oligomeric nucleus with a given size or a specific structure. According to the nucleation hypothesis, there can be many oligomeric species, with only a few leading to fibril formation. These oligomeric species are not well structurally-characterized.
Using multipe single-molecule FRET experiments and modelling, we aim at structurally characterizing species of alpha-Synuclein small oligomers that interact with membranes. The fine structure of these oligomeric species will aid in the rational design of small molecules that will inhibit these alpha-Synuclein oligomers from forming.
The pre-genetic mechanism of antibiotic resistance
antibiotic molecules induce a dual effect on their molecular targets: while they specifically bind to their target molecules (a specific bacterial protein) and inhibit its activity, they also induce an environmental response by the bacteria that eventually lead to mutations in the target proteins. In turn, natural selection of these mutations lead to ones that stay functional and are not inhibited by the antibiotic molecule. The question is, how does an antibiotic molecule that is supposed to stop DNA transcription, allow the bacteria to keep living enough to induce the environmental change leading to antibiotic resistance?
In acute conditions of Tuberculosis infection, antibiotic molecules from the Rifamycin family are used for the specific inhibition of DNA transcription in bacteria. These antibiotic molecules bind with high affinity to one structure of the transcription complex.
The transcription complex encompasses many different structures that are dynamically changing. Some of these structures allow transcriptional activity without the inhibition of Rifamycins. The problem is that these alternative dynamic structures are not abundant, and therefore were not characterized by classical techniques of structural biology. In my lab we will characterize these dynamic structures using the combination of Single-molecule fluorescence spectroscopy and computational simulations.
The aim: forming the missing structural infrastructure for the development of antibiotics that will minimize antibiotic resistance
The mechanism of allosteric signal transmission
protein systems function as autonomous nano-machines. One of their most fascinating capabilities is the transfer of a signal along the protein structure. In Allostery, the binding of a ligand in one protein site leads to a change in the affinity of a distant site for another ligand. The question is how?
We shall develop a method that allows the direct measurement of allosteric signal transmission in proteins acting as enzymes and as molecular machines, we will study the molecular determinants of allosteric signal transmission and we will use these principles to engineer proteins with designed allosteric signal transmission.
Allosteric signal transmission through DNA
One fascinating example of allostery as a means to communicate regulatory signals is when it is transmitted on top of DNA. More specifically, when DNA-binding proteins bind their specific site on DNA, the binding usually deforms DNA at that site. It has recently been shown that the binding of one DNA binding protein to its site on DNA, which induces a deformation in DNA, induces also changes in binding affinities of other DNA binding proteins to their relevant binding target apart from the deformed target by tens of base-pairs.
Some of these protein-induced DNA deformations are fluctuating between different structures. Do some of these deformational fluctuations migrate on top of DNA? Are they the source for allosteric transmission through DNA? Can allosteric signal trasmission on DNA be designed, hence used for applications?
From the biological side, I will study how protein bound at one site on DNA can affect the functionality of another protein bound to DNA at a remote site. Specifically I am interested in understanding whether such a mechanism can explain how one bound RNA polymerase may regulate “traffic” of other RNA polymerases on top of the same gene.
Development of methods for Biochemistry & Biophysics
In my laboratory we probe structural dynamics by using single-molecule fluorescence-based techniques, such as FRET, PIFE (protein-induced fluorescence enhancement), and we combine these experimental results with simulations of macromolecular structure & dynamics. To probe conformational dynamics of multiple distant sites, we will use multi-color FRET at the single-molecule level.
Additionally, we are always striving to get better in our ability to image single molecules in their most rare states.
FRET imaging of weak protein-protein interactions
One of the most common uses of the FRET <10 nm ruler is to identify protein-protein interactions in fluorescence imaging. Assume we suspect proteins A & B interact in the cell. Imaging them as they interact can be classically achieved by genetically fusing a fluorescent protein (FP) to protein A and another FP to protein B. The two FP's fused to proteins A & B will serve as donor & acceptor of FRET. This assay works well in the case of strong interactions between A & B. Many A & B proteins will be found together (in within the FRET distance range) for long periods of time. However, on the other hand, if the results of the fluorescence imaging do not indicate A & B are interacting, does it necessarily mean there are no pairs of interacting A & B's?
The case of weak interactions are a challenge in biology - we cannot infer from negative results of FRET imaging that there are necessarily no interactions. The best we can do is to claim that if there are interactions, there are just a few of them, and they are below the background. Indeed, if proteins A & B weakly interact, there will be short periods of time in which a few of them will be found together (in within the FRET distance range). This means that each voxel (3D pixel) of the microscopic image will include many proteins A & B that are not interacting, and maybe just a few that are interacting, so most of the signal will be of pure fluorescence with no signatures of FRET.
In the Lerner lab we are now developing a new imaging technique that will allow elucidating images just with the interacting proteins.