John M. Rosenberg

MULTIDIMENSIONAL FREE-ENERGY CALCULATIONS USING THE WEIGHTED HISTOGRAM ANALYSIS METHOD

The recently formulated weighted histogram analysis method (WHAM)1 is an extension of Ferrenberg and Swendsens multiple histogram technique for free‐energy and potential of mean force calculations. As an illustration of the method, we have calculated the two‐dimensional potential of mean force surface of the dihedrals gamma and chi in deoxyadenosine with Monte Carlo simulations using the all‐atom and united‐atom representation of the AMBER force fields. This also demonstrates one of the major advantages of WHAM over umbrella sampling techniques. The method also provides an analysis of the statistical accuracy of the potential of mean force as well as a guide to the most efficient use of additional simulations to minimize errors.

The Mechanism of Sodium and Substrate Release from the Binding Pocket of Vsglt

Membrane co-transport proteins that use a five-helix inverted repeat motif have recently emerged as one of the largest structural classes of secondary active transporters. However, despite many structural advances there is no clear evidence of how ion and substrate transport are coupled. Here we report a comprehensive study of the sodium/galactose transporter from Vibrio parahaemolyticus (vSGLT), consisting of molecular dynamics simulations, biochemical characterization and a new crystal structure of the inward-open conformation at a resolution of 2.7 Å. Our data show that sodium exit causes a reorientation of transmembrane helix 1 that opens an inner gate required for substrate exit, and also triggers minor rigid-body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose readily to enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium-binding sites, the unwound portion of transmembrane helix 1 and the substrate-binding site that is essential in the coupling of co-transport.

Structure and function of restriction endonucleases: Current Opinion in Structural Biology 1991, 1:104–113

Abstract The past year has seen significant advances in our understanding of the structure and function of restriction endonucleases. The highlights include a revised chain tracing for Eco RI endonuclease from Escherichia coli , structures soon to be reported for E. coli Eco RV endonuclease and significant advances in the biochemistry and molecular genetics of both enzymes.

Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases.

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.

Statistical methods for the objective design of screening procedures for macromolecular crystallization

The crystallization of a new macromolecule is still very much a trial-and-error process. As is well known, it requires the search of a large parameter space of experimental settings to find the relatively few idiosyncratic conditions that lead to diffraction-quality crystals. Crystallographers have developed a variety of screens to help identify initial crystallization conditions, including those based on systematic grids, incomplete factorial and sparse-matrix approaches. These are somewhat subjectively formulated based on accumulated data from past crystallization experiments. Ideally, one would prefer as objective a procedure as possible; however, that requires objective methods that incorporate a broad source of crystallization data. The Biological Macromolecular Crystallization Database (BMCD), a repository of all published crystallization conditions, is an obvious source of this data. This database has been augmented with a hierarchical classification of the macromolecules contained in the BMCD as well as extensive data on the additives used with them. A statistical analysis of the augmented BMCD shows the existence of significant correlations between families of macromolecules and the experimental conditions under which they crystallize. This in turn leads to a Bayesian technique for determining the probability of success of a set of experimental conditions based on the data in the BMCD as well as facts about a macromolecule known prior to crystallization. This has been incorporated into software that enables users to rank experimental conditions for new macromolecules generated by a dense partial factorial design. Finally, an additional advantage of the software described here is that it also facilitates the accumulation of the data required for improving the accuracy of estimation of the probabilities of success - knowledge of the conditions which lead to failure of crystallization.

Water Permeation Through the Sodium-Dependent Galactose Cotransporter VSGLT

It is well accepted that cotransporters facilitate water movement by two independent mechanisms: osmotic flow through a water channel in the protein and flow driven by ion/substrate cotransport. However, the molecular mechanism of transport-linked water flow is controversial. Some researchers believe that it occurs via cotransport, in which water is pumped along with the transported cargo, while others believe that flow is osmotic in response to an increase in intracellular osmolarity. In this letter, we report the results of a 200-ns molecular dynamics simulation of the sodium-dependent galactose cotransporter vSGLT. Our simulation shows that a significant number of water molecules cross the protein through the sugar-binding site in the presence as well as the absence of galactose, and 70-80 water molecules accompany galactose as it moves from the binding site into the intracellular space. During this event, the majority of water molecules in the pathway are unable to diffuse around the galactose, resulting in water in the inner half of the transporter being pushed into the intracellular space and replaced by extracellular water. Thus, our simulation supports the notion that cotransporters act as both passive water channels and active water pumps with the transported substrate acting as a piston to rectify the motion of water.

A Kinesin Switch I Arginine to Lysine Mutation Rescues Microtubule Function

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.

PCAM: a multi-user facility-based protein crystallization apparatus for microgravity

A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of the many important examples of quality improvements gained from crystal growth in the diffusion-limited environment in space. The concept was based on the adaptation for microgravity of a commonly available crystallization tray to increase sample density, to facilitate co-investigator participation and to improve flight logistics and handling. A co-investigator group representing scientists from industry, academia, and government laboratories has been established. Microgravity applications of the hardware have produced improvements in a number of structure-based crystallographic studies and include examples of enabling research. Additionally, the facility has been used to support fundamental research in protein crystal growth which has delineated factors contributing to the effect of microgravity on the growth and quality of protein crystals.

Cluster analysis of the Biological Macromolecule Crystallization Database

Abstract Cluster analysis was performed on the Biological Macromolecule Crystallization Database (BMCD) [1] in an effort to uncover trends useful in the crystallization of new macromolecules. The following crystallization parameters were used in defining an experiment; pH, temperature, molecular weight, macromolecular concentration, precipitant type and crystallization method. Using these parameters, a measure of the difference between experiments is developed. Groups or clusters of similar experiments are identified as those close together based upon the difference measure. Descriptive statistics were performed on each cluster. Since each cluster represents a well defined class of macromolecules, it seems reasonable to use the boundaries of conditions within each cluster as initial conditions when attempting to crystallize a new macromolecule.

Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT

Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.