The mechanical heterogeneity of biomolecular structures is intimately linked to their diverse biological functions. Applying rigidity theory to biomolecules identifies this heterogeneous composition of flexible and rigid regions, which can aid in the understanding of biomolecular stability and long-ranged information transfer through biomolecules, and yield valuable information for rational drug design and protein engineering. We review fundamental concepts in rigidity theory, ways to represent biomolecules as constraint networks, and methodological and algorithmic developments for analyzing such networks and linking the results to biomolecular function. Software packages for performing rigidity analyses on biomolecules in an efficient, automated way are described, as are rigidity analyses on biomolecules including the ribosome, viruses, or transmembrane proteins. The analyses address questions of allosteric mechanisms, mutation effects on (thermo-)stability, protein (un-)folding, and coarse-graining of biomolecules. We advocate that the application of rigidity theory to biomolecules has matured in such a way that it could be broadly applied as a computational biophysical method to scrutinize biomolecular function from a structure-based point of view and to complement approaches focused on biomolecular dynamics. We discuss possibilities to improve constraint network representations and to perform large-scale and prospective studies.

Improving an enzyme's (thermo-)stability or tolerance against solvents and detergents is highly relevant in protein engineering and biotechnology. Recent developments have tended toward data-driven approaches, where available knowledge about the protein is used to identify substitution sites with high potential to yield protein variants with improved stability, and subsequently, substitutions are engineered by site-directed or site-saturation (SSM) mutagenesis. However, the development and validation of algorithms for data-driven approaches have been hampered by the lack of availability of large-scale data measured in a uniform way and being unbiased with respect to substitution types and locations. Here, we extend our knowledge on guidelines for protein engineering following a datadriven approach by scrutinizing the impact of substitution sites on thermostability or/and detergent tolerance for Bacillus subtilis lipase A (BsLipA) at very large scale. We systematically analyze a complete experimental SSM library of BsLipA containing all 3439 possible single variants, which was evaluated as to thermostability and tolerances against four detergents under respectively uniform conditions. Our results provide systematic and unbiased reference data at unprecedented scale for a biotechnologically important protein, identify consistently defined hot spot types for evaluating the performance of data-driven protein-engineering approaches, and show that the rigidity theory and ensemble-based approach Constraint Network Analysis yields hot spot predictions with an up to ninefold gain in precision over random classification.

Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible.However, examples for the opposite received much less attention. Here, we exploit comprehensive experimental information on the substrate promiscuity of 147 esterases tested against 96 esters together with computationally efficient rigidity analyses to understand the molecular origin of the observed promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly less flexible than specific ones, are significantly more thermostable, and have a significantly increased specific activity. These results may be reconciled with a model according to which structural flexibility in the case of specific esterases serves for conformational proofreading. Our results signify that an esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.

34Understanding mechanisms of promiscuity is increasingly important from a fundamental and 35 application point of view. As to enzyme structural dynamics, more promiscuous enzymes 36 generally have been recognized to also be more flexible. However, examples for the opposite 37 received much less attention. Here, we exploit comprehensive experimental information on 38 the substrate promiscuity of 147 esterases tested against 96 esters together with 39 computationally efficient rigidity analyses to understand the molecular origin of the observed 40 promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly 41 less flexible than specific ones, are significantly more thermostable, and have a significantly 42 increased specific activity. These results may be reconciled with a model according to which 43 structural flexibility in the case of specific esterases serves for conformational proofreading. 44 Our results signify that esterase sequence space can be screened by rigidity analyses for 45 promiscuous esterases as starting points for further exploration in biotechnology and synthetic 46 chemistry. 47 48 3 1. Introduction 49 Enzymes involved in primary metabolism typically exquisitely discriminate against other 50metabolites. Yet, evolution of specificity is only pushed by nature to the point at which 51 'unauthorized' reactions do not impair the fitness of the organism (1). As a result, the universe 52 of promiscuous activities available in nature has been suggested to be enormous (2,3). 53 Understanding mechanisms of promiscuity has thus become increasingly important for the 54 fundamental understanding of molecular recognition and how enzyme function has evolved 55 over time(4) but also to optimize enzyme engineering applications (5). A particular challenge 56 in the latter case is the ability to discover a suitable enzyme with 'sufficient' promiscuous 57 activity to serve as a starting point for further exploration (1). 58Enzyme structural dynamics, besides its role in catalysis (6, 7) and allosteric regulation (8-59 11), has been recognized as likely the single most important mechanism by which promiscuity 60 can be achieved (5). Prominent examples are human cytochrome P450 (CYP) enzymes, for 61 which crystallographic studies and molecular simulations demonstrated that more 62 promiscuous CYPs show larger structural plasticity and mobility (12-14), or TEM-1 -63 lactamase and a resurrected progenitor, for which molecular simulations show that the pocket 64 of the ancestral, and more promiscuous, enzyme fluctuates to a greater extent (15). However, 65 examples for the opposite, i.e., conformational changes selected in evolution such that they 66 enhance specificity in molecular recognition (16), have received much less attention in the 67 context of enzyme promiscuity. 68A clear limitation for scrutinizing the link between enzyme structural dynamics and substrate 69 promiscuity is the general lack of large-scale data on one enzyme (super)family tested against 70 a multitude of ...

Background Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels.ResultsIn total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously.ConclusionsSingle amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0772-z) contains supplementary material, which is available to authorized users.